Targeted delivery of drugs, therapeutic nucleic acids and functional nucleic acids to mammalian cells via intact killed bacterial cells

ABSTRACT

A composition comprising intact killed bacterial cells that contain a therapeutic nucleic acid, a drug or a functional nucleic acid is useful for targeted delivery to mammalian cells. The targeted delivery optionally employs bispecific ligands, comprising a first arm that carries specificity for a killed bacterial cell surface structure and a second arm that carries specificity for a mammalian cell surface receptor, to target killed bacterial cells to specific mammalian cells and to cause endocytosis of the killed bacterial cells by the mammalian cells. Alternatively, the delivery method exploits the natural ability of phagocytic mammalian cells to engulf killed bacterial cells without the use of bispecific ligands.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/184,068, filed Jul. 15, 2011, which is a division of U.S. patentapplication Ser. No. 11/765,635, filed Jun. 20, 2007, which claims thebenefit priority of U.S. Provisional Patent Application No. 60/815,883filed on Jun. 23, 2006, and U.S. Provisional Patent Application No.60/909,078 filed on Mar. 30, 2007, both of which are hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to targeted delivery, by means of intactkilled bacterial cells, of bioactive molecules, including therapeuticnucleic acids, functional nucleic acids, drugs, peptides, proteins,carbohydrates and lipids, to mammalian host cells.

A number of hurdles continue to challenge targeted delivery of bioactivemolecules to mammalian cells (e.g., cancer cells), particularly in-vivo.Those hurdles include (a) composition, functional characteristics andstability of delivery vehicles, (b) packaging therapeuticallysignificant concentrations of bioactive molecules, (c) targeting desireddiseased cells in-vivo, (d) overcoming a series of intracellularbarriers and successfully deliver therapeutic concentrations ofbioactive molecules to intracellular targets, (e) avoiding a range ofhost immune elements such as antibodies, complement, and macrophagesthat may destroy a vector before it reaches a target, (f) crossing theendothelial barrier of blood vessel walls, particularly at the site of atumor mass, (g) migrating through several layers of cells to reach atarget (e.g., it is known that a solid tumor is an organized structurecontaining both tumor cells and normal cells; hence a vector must crossseveral layers of normal cells to access malignant cells), (h) migratingthrough an extracellular matrix (ECM) comprised of glycoproteins,sulfated glycosaminoglycans, hyaluronan, proteoglycans and collagen thatfills the space between cells and therefore hampers transport of avector, and (i) addressing high interstitial hypertension (elevatedhydrostatic pressure outside blood vessels) in the tumormicroenvironment, which may limit the access of bioactive molecules.

A number of different vectors have been proposed for both nucleic acidand drug delivery, including viral, non-viral non-living, and non-viralliving vectors. The non-viral non-living vectors have been adapted forboth nucleic acid and drug delivery. The other two types of vectors havebeen adapted for nucleic acid delivery. Non-viral living vectors aremainly being developed for direct tumor-cell killing capabilities. Whileall these vectors have advantages, they also have drawbacks.

Viral vectors, such as retrovirus, adenovirus, adeno-associated virus,pox virus, herpes simplex virus, and lentivirus, have been developed forgene delivery. However, viral vectors are unable to deliver genessystemically and specifically to primary and/or metastasized tumor cellswithout infecting normal tissues (Akporiaye and Hersh, 1999; Biederer etal., 2002; Green & Seymour, 2002). Additionally, the extremely limiteddiffusibility of virions within extracellular spaces significantlyhinders the dissemination of viral vectors. Moreover, viruses areantigenic, and therefore give rise to host immune responses. Such immuneresponses include both specific adaptive responses and non-specificinnate responses (Chen et al., 2003; Ferrari et al., 2003; Wakimoto etal., 2003). The latter plays an important role in eliminating adenoviralvectors (Liu and Muruve, 2003) and HSV (Wakimoto et al., 2003).

Non-viral non-living vectors are exemplified by cationic polymers(polyplexes), cationic lipids (liposomes, lipoplexes) and syntheticnanoparticles (nanoplexes). They are more versatile than viral vectors,and offer several distinct advantages because their molecularcomposition can be controlled, manufacturing and analysis of suchvectors is fairly simple, they can accommodate a range of transgenesizes (Kreiss et al., 1999; de Jong et al., 2001) and they are lessimmunogenic (Whitmore et al., 1999, 2001; Dow et al., 1999; Ruiz et al.,2001). The efficiency of gene delivery with non-viral non-living vectorsis significantly less, however, than with viral vectors. At least 10⁶plasmid copies are needed to transfect a single cell, with approximately10²-10⁴ copies actually making it to the nucleus for transgeneexpression (Feigner and Ringold, 1989; James and Giorgio, 2000;Tachibana et al., 2002). This inefficiency is attributable to theinability of non-viral non-living vectors to overcome the numerouschallenges encountered between a site of administration and localizationin a target cell nucleus, including, (a) the physical and chemicalstability of DNA and its delivery vehicle in the extracellular space,(b) cellular uptake by endocytosis, (c) escape from the endosomalcompartments prior to trafficking to lysosomes and cytosolic transport,and (d) nuclear localization of the plasmid for transcription. Inaddition to these physical and chemical obstacles, biological barriers,such as immunogenic responses to the vector itself and immunestimulation by certain DNA sequences containing a central unmethylatedCpG motif exist (Yew et al., 1999; Scheule, 2000; Ruiz et al., 2001).

As an alternate to non-living nucleic acid/drug delivery vehicles, livebacterial vectors have also been developed for tumor targeted therapy(Pawalek et al., 2003; Soghomonyan et al., 2005). These vectors do notcarry a payload of nucleic acids or drugs, but preferentially accumulatein tumor cells, replicate intracellularly and kill the infected cells(Pawelek et al., 1997). This phenomenon is thought to be facilitated bya complex bacterial system for introducing bacterial proteins directlyinto mammalian cells, which can result in the induction of apoptosis(Chen et al., 1996; Monack et al., 1996; Zhou et al., 2000). Currently,Bifidobacterium (Yazawa et al., 2000; 2001; Li et al., 2003),Clostridium (Minton et al., 1995; Fox et al., 1996; Lemmon et al., 1997;Theys et al., 2001; Dang et al., 2001; Nuyts et al., 2002a; 2002b; Liuet al., 2002) Salmonella (Pawelek et al., 1997; Low et al., 1999; Plattet al., 2000; Luo et al., 2001; Rosenberg et al., 2002) and Vibrio (Yuet al., 2004) are under investigation as tumor-selective live bacterialvectors.

Live attenuated bacteria have also been explored as vehicles fordelivering nucleic acids (Paglia et al., 2000; Weiss and Chakraborty2001; Yuhua et al., 2001), which may encode angiogenic inhibitors (Leeet al. 2005a; 2005b; Li et al., 2003), prodrug-converting enzymes (Kinget al., 2002) or cytokines (Yamada et al., 2000). Significant drawbacksof this approach include (a) live recombinant bacteria gradually loseplasmid DNA in vivo, mainly due to the absence of selection pressure andassociated plasmid segregation, (b) bacteria carrying plasmid DNA tendto have a lower growth rate and appear to accumulate at lower levels andreside for a shorter period of time within tumors than bacteria withoutplasmids, (c) live Gram-negative bacterial vectors can cause severeendotoxin response in mammalian hosts, possibly due to in-vivo sheddingof endotoxin (lipopolysaccharide; LPS), and evoke a Toll-like receptorresponse due to cellular invasion, (d) most of the tumor-targeting livebacteria accumulate and grow in the necrotic and relatively hypoxic fociwithin tumors, but not in well-oxygenated tumors at the rim of thegrowing nodules where tumor cells are normally most aggressive, (e) therisk associated with possible reversion to a virulent phenotype of thesebacteria is a major concern (Dunham. 2002), and (f) the risk ofinfecting normal cells may lead to bacteremia and associated septicshock. The latter may particularly be a problem in immuno-compromisedpatients, such as late stage cancer patients.

Because problems continue to hamper the success of cancer therapeuticsin particular, an urgent need exists for targeted delivery strategiesthat will either selectively deliver bioactive agents to tumor cells andtarget organs, or protect normal tissues from administeredantineoplastic agents. Such strategies should improve the efficacy oftreatment by increasing the therapeutic indexes of anticancer agents,while minimizing the risks of therapy-related toxicity.

The present invention provides a versatile delivery vehicle for improveddrug, therapeutic nucleic acid and functional nucleic acid deliverystrategies, especially but not exclusively in the context of cancerchemotherapy.

SUMMARY OF THE INVENTION

To address these and other needs, the present invention provides, in oneaspect, a composition that comprises a plurality of intact killedbacterial cells and a pharmaceutically acceptable carrier. The killedbacterial cells contain a therapeutic nucleic acid, a drug or afunctional nucleic acid. With respect to the latter, in one embodimentthe functional nucleic acid is plasmid-free. In this regard, functionalnucleic acids are packaged directly into killed bacterial cells bypassing through the bacterial cell's intact membrane, without usingplasmid-based expression constructs or the expression machinery of ahost cell. Such plasmid-free functional nucleic acids are exemplified bysingle-, double-, or multi-stranded DNA or RNA. In one embodiment,killed bacterial cells contain plasmid-free functional nucleic acid thatis regulatory RNA. In a preferred embodiment, the composition isessentially free of endotoxin.

The invention also provides bispecific ligands useful for targetingkilled bacterial cells to mammalian host cells. The bispecific ligandmay be polypeptide, carbohydrate or glycopeptide, and may comprise anantibody or antibody fragment. In preferred embodiments, the bispecificligand has a first arm that carries specificity for a bacterial surfacestructure and a second arm that carries specificity for a mammalian cellsurface structure. Further, the first arm and the second arm of thebispecific ligand may be monospecific or multivalent. A desirablebacterial surface structure for ligand binding is an O-polysaccharidecomponent of a lipopolysaccharide (LPS). Desirable mammalian cellsurface structures for ligand binding are receptors, preferably thosecapable of activating receptor-mediated endocytosis.

According to another aspect, the invention provides a delivery methodthat comprises bringing a plurality of killed bacterial cells intocontact with mammalian cells that are phagocytosis- orendocytosis-competent, such that the killed bacterial cells are engulfedby the mammalian cells and release their payload intracellularly. Thepayload may comprise a therapeutic nucleic acid, a functional nucleicacid or a drug.

In one embodiment, a method of delivering a functional nucleic acid,comprises (a) providing a plurality of killed bacterial cells in apharmaceutical carrier, each killed bacterial cell of the pluralityencompassing (i) a functional nucleic acid or (ii) a plasmid comprisedof a segment that encodes a functional nucleic acid and then (b)bringing said killed bacterial cells of the plurality into contact withtarget mammalian cells, such that said mammalian cells engulf saidkilled bacterial cell, whereby said functional nucleic acid is releasedinto the cytoplasm of the target cell. In one aspect, the killedbacterial cells are plasmid-free, while in another the functionalnucleic acid is regulatory RNA.

According to another aspect, the invention provides a targeted deliverymethod that comprises bringing bispecific ligands into contact with (i)intact killed bacterial cells that contain a desired payload and (ii)mammalian cells, preferably non-phagocytic mammalian cells. Thebispecific ligands have specificity for both a surface component on theintact killed bacterial cells and a surface component on the mammaliancells, such as a receptor. As a result, the ligands cause the killedbacterial cell to bind to the mammalian cells, the killed bacterialcells are engulfed by the mammalian cells, and the payload contained inthe killed bacterial cells is released into the cytoplasm of themammalian cell. The payload may comprise a therapeutic nucleic acid, afunctional nucleic acid or a drug.

In yet another aspect, the invention provides a method of overcomingdrug resistance or apoptosis resistance and treating a malignancy in asubject by delivering a functional nucleic acid to a target cell. Themethod comprises bringing a killed bacterial cell that contains (i) afunctional nucleic acid molecule or (ii) a plasmid comprised of asegment that encodes a functional nucleic acid molecule into contactwith a target mammalian cell. The mammalian cell engulfs the killedbacterial cell, the functional nucleic acid is released into thecytoplasm, transported to the nucleus and expressed by the target cell.

In relation to this invention, the contact between killed bacterialcells and mammalian cells may be in vitro or in vivo.

The invention further provides methods for loading killed bacterialcells with a drug. One such method involves creating a concentrationgradient of the drug between an extracellular medium containing thekilled bacterial cells and the killed bacterial cell cytoplasm. The drugnaturally moves down this concentration gradient, into the killedbacterial cell cytoplasm. Leakage of the drug from the bacterialcytoplasm is prevented due to the bacterial cells being metabolicallyinactive.

Another method of loading killed bacterial cells with a drug involvesculturing a bacterial cell under conditions, such that the bacterialcell transcribes and translates a therapeutic nucleic acid encoding thedrug, such that the drug is released into the cytoplasm of the bacterialcell, and then killing the bacterial cell to form one or more killedbacterial cells containing the drug in their cytoplasm.

In accordance with another aspect, the present invention contemplates amethod for formulating a killed bacterial cell with a plasmid-freefunctional nucleic acid. The method comprises co-incubating a pluralityof killed bacterial cells with a functional nucleic acid, such asregulatory RNA like siRNA, miRNA or shRNA, in a buffer. In someembodiments, the co-incubation may involve gentle shaking, while inothers the co-incubation is static. In some aspects, the co-incubationlasts about half an hour, while in others it lasts about an hour. In oneembodiment, the buffer comprises buffered saline, for example, a 1×phosphate buffer solution. In another embodiment, the co-incubation isconducted at a temperature of about 4° C. to about 37° C., about 20° C.to about 30° C., about 25° C., or about 37° C. The co-incubation cancomprise about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or 10¹³ killed bacterialcells.

The present invention contemplates a use of intact killed bacterialcells and bispecific ligands in the preparation of a medicament, for usein a method of treating a disease or modifying a trait by administrationof the medicament to a cell, tissue, or organ. In the medicament, thekilled bacterial cells contain a therapeutic nucleic acid molecule, adrug or a functional nucleic acid molecule, and, optionally, bispecificligands that are capable of binding to the killed bacterial cells and totarget non-phagocytic mammalian cells. Such medicaments are useful totreat various conditions and diseases by increasing expression orfunction of a desired protein, or by inhibiting expression or functionof a target protein. Illustrative of such conditions and diseases are acancer and an acquired disease, such as AIDS, pneumonia emphysema, andtuberculosis. Alternatively, the treatment may affect a trait, such asfertility, or an immune response associated with an allergen or aninfectious agent.

The present invention also provides a pharmaceutically acceptable methodfor purifying intact killed bacterial cells. The method combines (i)killing live bacterial cells with antibiotics, (ii) cross-flowfiltration and/or dead-end filtration, to eliminate free endotoxin,cellular debris, free nucleic acids, bacterial membrane blebs, mediacontaminants, and (iii) antibody-based sequestration, to eliminateresidual free endotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows highly significant anti-tumor effects via bispecificantibody-targeted, chemotherapeutic drug-packaged intact killedbacterial cells. Human breast cancer (MDA-MB-468) xenograft wasestablished subcutaneously (between the shoulder blades) in Balb/c nu/numice and when tumor volumes reached ˜70 mm³, mice were treated (n=11mice per group) intravenously (i.v.) with free doxorubicin (G2), ornon-targeted intact killed S. Typhimurium bacterial cells packaged withdox (G3), or with EGFR-targeted intact killed S. Typhimurium bacterialcells packaged with dox (G4). G1 mice were controls and received sterilephysiological saline (i.v.). The treatments were administered on thedays marked by a triangle on the x-axis and tumor volume was measured asshown on the y-axis. The result shows a highly significant anti-tumoreffect when ^(EGFR)killed S. typhimurium _(Dox) (G4) was used as atreatment while the G2 and G3 mice showed no anti-tumor effects.Standard deviation is shown for each measurement.

FIG. 2 graphically shows that intact killed bacterial cells packagedwith paclitaxel or siRNA inhibit the growth of human colon cancer cell(HCT116) tumors in vivo.

FIG. 3 shows the reversal of drug resistance in mice carrying humancolon cancer (Cato-2) xenografts by using a dual treatment protocol,wherein the first treatment comprises EGFR-packaged, killed S.typhimurium carrying anti-MDR-1 shRNA and the second treatment comprisesEGFR-packaged, killed S. typhimurium carrying either Irinotecan or5-fluorouracil (5-FU). The first treatment and second treatments areshown by a triangle and an arrow, respectively, below the x-axis.

DETAILED DESCRIPTION

The present inventors have determined that intact killed bacterial cellsare effective vehicles for targeted delivery of therapeutic nucleicacids, functional nucleic acids and drugs to diseased cells,particularly cancer cells, both in vitro and in vivo. A number ofsurprising discoveries underlie that determination.

For example, the inventors discovered that when compositions comprising(a) intact killed bacterial cells containing a therapeutic nucleic acid,drug or functional nucleic acid payload (b) bispecific targetingligands, and (c) a pharmaceutically acceptable carrier are brought intocontact with diseased cells in vitro or in vivo, the intact killedbacterial cell vehicles are endocytosed at high efficiency into targetnon-phagocytic mammalian cells. This discovery was a surprise, becausealthough bispecific ligands have been used to target viral and non-viraldelivery vehicles to non-phagocytic mammalian cells (Wickham et al.,1996; Nettelbeck et al., 2001; Boucher et al., 2003; Ogris & Wagner,2002), it was believed that receptor-mediated endocytosis would not workfor particles as large as bacterial cells.

For instance, adenoviral vectors have been redirected to targetmammalian cell-surface receptors, such as endoglin on endothelial cells,and internalized via clathrin-coated pits in the mammalian cell plasmamembrane. Wickham et al., 1996; Nettelbeck et al., 2001; Boucher et al.,2003. The clathrin-coated pits resemble a cup that envelopes the vector,but the size of the cup is understood to be a limiting factor.Clathrin-coated pits have a limited size of 85-110 nm, due to the sizeof the clathrin coat. Swanson & Watts, 1995. Bacterial cells, bycontrast, are at least 400 nm in diameter and 1,000 nm in length. Hence,is was not expected that such a targeting approach would work for killedbacterial cells.

Knowledge concerning other large vectors supported the expectation thatkilled bacterial cells would not be internalized through clathrin-coatedpits. For instance, large lipoplexes (non-viral vectors up to 500 nm)preferentially enter cells by receptor- and clathrin-independentendocytosis, while smaller lipoplexes (less than 200 nm) can beinternalized via a non-specific, clathrin-dependent process. Simoes etal., 1999. Likewise, large viruses, such as vaccinia virus, on the orderof 350 nm×250 nm in size, do not infect mammalian cells via aclathrin-coated pathway. Essani and Dales, 1979.

In a similar vein, non-phagocytic mammalian cells cannot engulf largepathogens, like bacterial cells. Only professional phagocytes likemacrophages engulf such pathogens, and the engulfment process isclathrin- and receptor-independent, being accomplished by phagocytosis.The interaction of large pathogens with the cell surface induces acomplex signaling cascade, leading to actin rearrangements at the plasmamembrane to form a large phagocytic cup, which engulfs the bacterium.Dramsi and Cossart, 1998. The signaling cascades that are responsible,on bacterial entry, for actin rearrangements at the plasma membrane arepoorly understood. Galan, 1996; Menard et al., 1996; Finlay and Cossart,1997; Dramsi and Cossart, 1998.

Specific investigations into the effect of particle size onreceptor-mediated endocytosis show that the process is stronglysize-dependent. For example, Aoyama et al., 2003, studied the effect ofparticle size on glycoviral gene delivery and concluded that the optimalparticle size for receptor-mediated endocytosis is ˜25 nm. See alsoNakai et al., 2003; Osaki et al., 2004. Gao et al., 2005, confirmed thatconclusion.

Moreover, even though bispecific ligands reportedly have been used tore-direct viral vectors, the method has not always been successful inthe context of gene delivery. In attempted retargeting of viruses fromtheir native receptors to alternative receptors, many experiments haveshown that cell surface attachment is insufficient for sustained viralentry and gene expression. Also, when virus envelope proteins weremodified for re-targeting, they exhibited low fusion activity, resultingin inefficient viral entry into cells. Zhao et al., 1999. In the absenceof specific targeting, strategies have depended on direct injection to alocalized site. Akporiaye & Hersh, 1999.

Thus, the art suggested that bispecific ligands would not enable intactkilled bacterial cell vehicles to enter non-phagocytic mammalian cells.In further support of this point, the inventors discovered thatnon-targeted killed bacterial cells are unable to specifically adhere toand deliver a payload to non-phagocytic mammalian cells, even afterrepeated attempts with prolonged incubation periods in a number ofmammalian cell lines. In particular, non-targeted killed bacterial cellsare not internalized by non-phagocytic mammalian cells. By contrast,killed bacterial cells are readily phagocytosed by professionalphagocytes like macrophages. This corroborates earlier findings thatmicroparticles up to 12 μm are phagocytosed by professional phagocytes(Kanke et al., 1983) and that maximal uptake of microparticles intomacrophages occurs with particles of <2 μm (Tabata and Ikada, 1988;1990). Unlike viral vectors that specifically adhere to viral receptorsand trigger their internalization, therefore, killed bacterial cellshave no similar mechanism to invade enter non-phagocytic mammaliancells.

Against this background, the inventors also discovered that bispecificligands can direct the endocytosis of intact killed bacterial cellswithin non-phagocytic mammalian cells. Preliminary data suggests thatinternalization of bacterial cells may occur via the receptor- andmacropinocytosis-dependent pathway, though the Applicants are not boundto such a theory.

The inventors further discovered that following endocytosis, the killedbacteria are completely degraded in intracellular vacuoles, presumablyendo-lysosomal compartments. This was surprising because harshdegradative mechanisms that are capable of degrading large biologicalparticles like bacterial and parasitic cells were thought to operateonly in professional phagocytes, like macrophages. Those mechanisms werethought to permit full antigen processing and presentation byprofessional phagocytes. Because most non-phagocytic cells do notprocess and present antigens, it was believed that they contained onlymild antigen processing systems that are mainly used for re-cycling ofcellular components.

After being internalized by receptor-mediated endocytosis, vectors areenclosed within endosomal or lysosomal membranes, and are thereforeseparated from the cytoplasm. This constitutes a significant impedimentto payload delivery, especially because endosomal and lysosomalcompartments can become highly caustic and degrade more than 99% of apayload, such as nucleic acids in a vector. Successful gene deliveryvectors have mechanisms that allow nucleic acids to enter the cytoplasm,but skilled artisans would not expect minicells to have such mechanisms.

Viruses, for example, have evolved sophisticated processes to enter themammalian cell cytoplasm. Enveloped retro-viruses, such as HIV-1, gainaccess to the cytoplasm by direct fusion with the plasma membrane. Steinet al., 1987. Non-enveloped viruses use various strategies to penetratethe endosomal membrane after endocytosis. For example, influenza virusesinduce fusion of the viral and endosomal membranes, which is triggeredby the acidic environment of the endosome. Marsh & Helenius, 1989. Atlow pH, the predominant influenza viral envelope glycoproteinhemagglutinin (HA) undergoes conformational changes, leading to theprotrusion of a hydrophobic spike into the endosomal membrane thatinitiates membrane fusion. Bullough et al., 1994. Adenoviruses also arebelieved to escape into the cytosol by a mechanism tied to acidificationof the endosome. Low pH has several effects on the adeno viral capsid.For example, the capsid's penton protein undergoes conformationalchanges that expose hydrophobic regions for endosomal membraneinteraction. Seth et al., 1985. Additionally, intrinsic proteaseactivity of the adeno viral capsid also seems to contribute to endosomalescape. Greber et al., 1996.

For liposomal vectors, the endosomal membrane barrier continues to limitthe efficiency of gene delivery. Successful release of liposomal nucleicacids is understood to result from disruption of the endo-lysosomemembrane. Xu & Szoka, 1996; El Ouahabi et al., 1997; Zelphati & Szoka,1996a; Wattiaux et al., 2000. Disruption of the endo-lysosomal membraneis thought to occur via transbilayer flip-flop of lipids, leading tomembrane destabilization and penetration of naked DNA into thecytoplasm. Zelphati & Szoka, 1996a; 1996b; Mui et al., 2000. Studieshave further demonstrated that cytoplasmic release of liposomal contentsinvolves (a) charge neutralization of a cationic complexing agent withanionic macromolecules such as anionic lipids and proteoglycans, (b)cationic lipid-mediated fusion, and (c) membrane destabilization bypH-sensitive lipids. Wrobel & Collins, 1995; Meyer et al., 1997; Clark &Hersh, 1999. Additional studies have shown that a mixture of neutrallipid (DOPE) with cationic lipid facilitates membrane disruption andincreases the amount of liposomal contents released into the cytoplasm,because DOPE promotes the fusion of liposome particles with endosomalmembranes. Farhood et al., 1995; Fasbender et al., 1997; Hafez et al.,2001. Also, cationic PEI and polyamine dendrimers have been used tofacilitate disruption of the endolysosomal membrane, because they havean extensive buffering capacity that provokes swelling and disruption ofendosomes. Klemm, 1998; Sonawane et al., 2003. Additional functionalitycan be incorporated into liposome vectors in the form of anendosomolytic pore forming protein from Listeria monocytogenes,listeriolysin O (LLO). Lorenzi and Lee, 2005. LLO is capable ofbreaching the endosomal membrane, thereby allowing escape of endosomalcontents into the cytoplasm. Lee et al., 1996.

Thus, current teachings suggest that sophisticated mechanisms arenecessary to allow some vector payload to escape the lysosomal membrane.The killed bacterial cell is a non-living particle and does not carryany lysosomal membrane destabilizing functions. The inventorsdiscovered, that if killed bacterial cells carry at least 70 to 100copies of plasmid DNA, then some of this DNA can escape the endosomalmembrane without the need to destabilize or disrupt the endosomalmembrane. This suggests that while most of the plasmid DNA is likely tobe degraded in the endo-lysosomal vacuole, it was possible to overwhelmthe system and thereby permit some DNA to escape intact into themammalian cell cytoplasm. Additionally, the inventors discovered thatalthough non-phagocytic mammalian cells are not thought to carry harshlysosomal processing mechanisms that could degrade complexmulti-component structures like bacterial cells, that may not be true.The current view specifies that intracellular degradation of suchcomplex structures like bacterial cells is limited to professionalphagocytic cells that are capable of complete antigen processing andpresentation.

In a related aspect, the inventors discovered that a significantconcentration of bioactive drug carried by bispecific ligand-targeted,drug-packaged killed bacterial cells also escapes the endo-lysosomalmembrane and enters the mammalian cell cytoplasm. Additionally, theydiscovered that killed bacterial cells are highly versatile in theircapacity to package a range of different drugs (e.g., hydrophilic,hydrophobic, and amphipathic drugs such as doxorubicin, paclitaxel,cisplatin, carboplatin, 5-fluorouracil, and irinotecan) and have foundthat all are readily packaged in killed bacterial cells intherapeutically significant concentrations.

The inventors further discovered that when bispecific antibody-targeted,drug-packaged killed bacterial cells (for simplicity, also designated“therapeutic”) were administered intravenously into nude mice carryinghuman tumor xenografts, they extravasated from the blood vessel wallssurrounding the tumor mass and entered into the tumor microenvironment.

Targeting of particle-based systems in the context of cancer therapy hasexploited the leaky tumor vasculature (Jain, 1998) and the lack of aneffective lymphatic drainage (Maeda and Matsumura, 1989; Seymour, 1992;Yuan et al., 1994), which results in enhanced permeability and retention(EPR) effect (Maeda, 2001) of circulating particles (passive targeting).Tumor vessels have an irregular diameter, an abnormal branching pattern,and do not fit well into the usual classification of arterioles,capillaries, or venules. Warren, 1979; Less et al., 1991, 1997;Konerding et al 1995. Of particular functional importance, tumor vesselsare unusually leaky. Peterson and Appelgren, 1977; Gerlowski and Jain,1986; Jain, 1987, 1997; Dvorak et al., 1988. The hyperpermeability oftumor microvessels to large molecules has been observed in numerousstudies. Gerlowski and Jain, 1986; Jain, 1987; Jain 1996. However, theupper size limit for agents that can traverse vessels of differenttumors and how that is regulated are poorly understood. One studymeasured the pore cutoff size of a human colon carcinoma grownsubcutaneously in immunodeficient mice to be between 400-600 nm. Yuan etal., 1995. Others reported that some tumors have a pore cut-off size ofonly 100 nm. Hobbs et al., (1998). Accordingly, it was surprising tofind that intact killed bacterial cells larger than 1,000 μm are able toextravasate the endothelial cell wall surrounding tumors. This discoveryenables the use of intact killed bacterial cells for tumor therapy inviva.

Additionally, it previously was suggested that the abnormal tumormicroenvironment is characterized by interstitial hypertension (elevatedhydrostatic pressure outside the blood vessels; Less et al., 1992; Jain,2001) that limits access of anti-cancer therapeutics. For instance, itwas reported that when breast cancer (MDA-MD-231) tumors establishedorthotopically in SCID mice were studied following intravenous injectionof contrast agent Gadolinium diethylenetriamine-penta-acetate, there wasa decrease in the entry of the contrast agent to the tumor. Dadiani etal., 2004. The authors of that report speculated that the observedincrease in interstitial hypertension suggests that the highinterstitial pressure forces fluid to reenter the blood vessels, therebyincreasing outflux to influx ratio. Interestingly, the inventorsdiscovered that killed bacterial cells are not hindered by suchinterstitial hypertension, but are able to achieve highly significantanti-tumor effects (FIG. 1).

The following description outlines the invention related to thesediscoveries, without limiting the invention to the particularembodiments, methodology, protocols or reagents described. Likewise, theterminology used herein describes particular embodiments only, and doesnot limit the scope of the invention. Unless defined otherwise, alltechnical and scientific terms used in this description have the samemeaning as commonly understood by those skilled in the relevant art.Additionally, the singular forms “an,” and “the” include pluralreference unless the context clearly dictates otherwise.

Compositions Comprising Intact Killed Bacterial Cells

In one aspect, the invention provides a composition comprising intactkilled bacterial cells and a pharmaceutically acceptable carriertherefor. The killed bacterial cells may contain a therapeutic nucleicacid, a drug, a functional nucleic acid molecule or a combinationthereof.

Intact Killed Bacterial Cells

According to the invention, killed bacterial cells are non-livingprokaryotic cells of bacteria, cyanobateria, eubacteria andarchaebacteria, as defined in the 2nd edition of BERGEY'S MANUAL OFSYSTEMATIC BIOLOGY. Such cells are deemed to be “intact” if they possessan intact cell wall and/or cell membrane and contain genetic material(nucleic acid) that is endogenous to the bacterial species.

Therapeutic Nucleic Acids and Therapeutic Expression Products

A therapeutic nucleic acid molecule encodes a product, such as apeptide, polypeptide or protein, the production of which is desired in atarget cell. For example, the genetic material of interest can encode ahormone, receptor, enzyme, or (poly) peptide of therapeutic value. Suchmethods can result in transient expression of non-integrated transferredDNA, extrachromosomal replication and expression of transferredreplicons such as episomes, or integration of transferred geneticmaterial into the genomic DNA of host cells.

The phrase “nucleic acid molecules” and the term “polynucleotides”denote polymeric forms of nucleotides of any length, eitherribonucleotides or deoxynucleotides. They include single-, double-, ormulti-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. The backbone of a polynucleotide can comprise sugarsand phosphate groups, as is typical for RNA and DNA, or modified orsubstituted sugar or phosphate groups. Alternatively, the backbone ofthe polynucleotide can comprise a polymer of synthetic subunits such asphosphoramidites and thus can be an oligodeoxynucleoside phosphoramidateor a mixed phosphoramidate-phosphodiester oligomer. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs, uracyl, other sugars, and linking groups such asfluororibose and thioate, and nucleotide branches. A polynucleotide maybe further modified, such as by conjugation with a labeling component.Other types of modifications include caps, substitution of one or moreof the naturally occurring nucleotides with an analog, and introductionof means for attaching the polynucleotide to proteins, metal ions,labeling components, other polynucleotides, or a solid support.

“Polypeptide” and “protein,” used interchangeably herein, refer to apolymeric form of amino acids of any length, which may includetranslated, untranslated, chemically modified, biochemically modified,and derivatized amino acids. A polypeptide or protein may be naturallyoccurring, recombinant, or synthetic, or any combination of these.Moreover, a polypeptide or protein may comprise a fragment of anaturally occurring protein or peptide. A polypeptide or protein may bea single molecule or may be a multi-molecular complex. In addition, suchpolypeptides or proteins may have modified peptide backbones. The termsinclude fusion proteins, including fusion proteins with a heterologousamino acid sequence, fusions with heterologous and homologous leadersequences, with or without N-terminal methionine residues,immunologically tagged proteins, and the like.

The term “expression” generally refers to the process by which apolynucleotide sequence undergoes successful transcription andtranslation such that detectable levels of the amino acid sequence orprotein are expressed. In certain contexts herein, expression refers tothe production of mRNA. In other contexts, expression refers to theproduction of protein.

Transcription or translation of a given therapeutic nucleic acidmolecule may be useful in treating cancer or an acquired disease, suchas AIDS, pneumonia, emphysema, or in correcting inborn errors ofmetabolism, such as cystic fibrosis. Transcription or translation of atherapeutic nucleic acid may also effect contraceptive sterilization,including contraceptive sterilization of feral animals.Allergen-mediated and infectious agent-mediated inflammatory disordersalso can be countered by administering, via the present invention, atherapeutic nucleic acid molecule that, upon expression in a patient,affects immune response(s) associated with the allergen and infectiousagent, respectively. A therapeutic nucleic acid molecule also may havean expression product, or there may be a downstream product ofpost-translational modification of the expression product, that reducesthe immunologic sequalae related to transplantation or that helpsfacilitate tissue growth and regeneration.

The terms “Cancer,” “neoplasm,” “tumor,” “malignancy” and “carcinoma,”used interchangeably herein, refer to cells or tissues that exhibit anaberrant growth phenotype characterized by a significant loss of controlof cell proliferation. The methods and compositions of this inventionparticularly apply to precancerous, malignant, pre-metastatic,metastatic, and non-metastatic cells.

A therapeutic nucleic acid molecule may be the normal counterpart of agene that expresses a protein that functions abnormally or that ispresent in abnormal levels in a disease state, as is the case, forexample, with the cystic fibrosis transmembrane conductance regulator incystic fibrosis (Kerem et al., 1989; Riordan et al., 1989; Rommens etal., 1989), with ß-globin in sickle-cell anemia, and with any ofα-globin, ß-globin and γ-globin in thalassemia. Thus, an excessproduction of α-globin over ß-globin which characterizes ß-thalassemiacan be ameliorated by gene therapy, in accordance with the presentinvention, using an intact killed bacterial cell engineered to contain aplasmid incorporating a sequence that has an antisense RNA transcriptvis-à-vis a target sequence of the α-globin mRNA.

In the treatment of cancer, a therapeutic nucleic acid molecule suitablefor use according to the present invention could have a sequence thatcorresponds to or is derived from a gene that is associated with tumorsuppression, such as the p53 gene, the retinoblastoma gene, and the geneencoding tumor necrosis factor. A wide variety of solid tumors—cancer,papillomas, and warts—should be treatable by this approach, pursuant tothe invention. Representative cancers in this regard include coloncarcinoma, prostate cancer, breast cancer, lung cancer, skin cancer,liver cancer, bone cancer, ovary cancer, pancreas cancer, brain cancer,head and neck cancer, and lymphoma. Illustrative papillomas are squamouscell papilloma, choroid plexus papilloma and laryngeal papilloma.Examples of wart conditions are genital warts, plantar warts,epidermodysplasia verruciformis, and malignant warts.

A therapeutic nucleic acid molecule for the present invention also cancomprise a DNA segment coding for an enzyme that converts an inactiveprodrug into one or more cytotoxic metabolites so that, upon in vivointroduction of the prodrug, the target cell in effect is compelled,perhaps with neighboring cells as well, to commit suicide. Preclinicaland clinical applications of such a “suicide gene,” which can be ofnon-human origin or human origin, are reviewed by Spencer (2000),Shangara et al. (2000) and Yazawa et al. (2002). Illustrative of suicidegenes of non-human origin are those that code for HSV-thymidinekinase(tk), cytosine deaminase (CDA)+uracil phosphoribosyl-transferase,xanthine-guanine phosphoribosyl-transferase (GPT), nitroreductase (NTR),purine nucleoside phosphorylase (PNP, DeoD), cytochrome P450 (CYP4B1),carboxypeptidase G2 (CPG2), and D-amino acid oxidase (DAAO),respectively. Human-origin suicide genes are exemplified by genes thatencode carboxypeptidase A1 (CPA), deoxycytidine kinase (dCK), cytochromeP450 (CYP2B 1,6), LNGFR/FKBP/Fas, FKBP/Caspases, and ER/p53,respectively.

A suicide-gene therapy could be applied to the treatment of AIDS. Thisstrategy has been tested with suicide vectors that express a toxic geneproduct as soon as treated mammalian cells become infected by HIV-1.These vectors use the HIV-1 regulatory elements, Tat and/or Rev, toinduce the expression of a toxic gene such as α-diphtheria toxin,cytosine deaminase, or interferon-a2 after infection by HIV-1. SeeCuriel et al., 1993; Dinges et al., 1995; Harrison et al., 1992a;Harrison et al., 1992b; Ragheb et al., 1999.

The therapeutic nucleic acid of the invention typically is contained ona plasmid within the killed bacterial cell. The plasmid also may containan additional nucleic acid segment that functions as a regulatoryelement, such as a promoter, a terminator, an enhancer or a signalsequence, and that is operably linked to the therapeutic nucleic acidsegment. A suitable promoter can be tissue-specific or eventumor-specific, as the therapeutic context dictates.

The therapeutic nucleic acid may encode a suicide gene or a normalcounter part of a gene that expresses a protein that functionsabnormally or is present in abnormal levels in the mammalian cell.Moreover, the therapeutic nucleic acid may be contained on a plasmidcomprised of multiple nucleic acid sequences. Further, the plasmid maycontain a regulatory element and/or a reporter element.

The term “gene” refers to a polynucleotide sequence that comprisescontrol and coding sequences necessary for the production of apolypeptide or precursor. The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence. A genemay constitute an uninterrupted coding sequence or it may include one ormore introns, bound by the appropriate splice junctions. Moreover, agene may contain one or more modifications in either the coding or theuntranslated regions that could affect the biological activity or thechemical structure of the expression product, the rate of expression, orthe manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions, and substitutions ofone or more nucleotides. In this regard, such modified genes may bereferred to as “variants” of the “native” gene.

The term “host cell” refers to a cell that may be, or has been, used asa recipient for a recombinant vector or other transfer ofpolynucleotides, and includes the progeny of the original cell that hasbeen transfected. The progeny of a single cell may not necessarily becompletely identical in morphology or in genomic or total DNA complementas the original parent due to natural, accidental, or deliberatemutation.

Regulatory Elements

A nucleic acid molecule to be introduced via the approach of the presentinvention also can have a desired encoding segment linked operatively toa regulatory element, such as a promoter, a terminator, an enhancerand/or a signal sequence. A suitable promoter can be tissue-specific oreven tumor-specific, as the therapeutic context dictates.

A promoter is “tissue-specific” when it is activated preferentially in agiven tissue and, hence, is effective in driving expression, in thetarget tissue, of an operably linked structural sequence. The categoryof tissue-specific promoters includes, for example: thehepatocyte-specific promoter for albumin and a₁-antitrypsin,respectively; the elastase I gene control region, which is active inpancreatic acinar cells; the insulin gene control region, active inpancreatic beta cells; the mouse mammary tumor virus control region,which is active in testicular, breast, lymphoid and mast cells; themyelin basic protein gene control region, active in oligodendrocytecells in the brain; and the gonadotropic releasing hormone gene controlregion, which is active in cells of the hypothalamus. See Frain et al.(1990), Ciliberto et al. (1985), Pinkert et al., (1987), Kelsey et al.(1987), Swift et al. (1984), MacDonald (1987), Hanahan, (1985), Leder etal. (1986), Readhead et al. (1987), and Mason et al. (1986).

There also are promoters that are expressed preferentially in certaintumor cells or in tumor cells per se, and that are useful for treatingdifferent cancers in accordance with the present invention. The class ofpromoters that are specific for cancer cells is illustrated by: thetyrosinase promoter, to target melanomas; the MUCl/Df3 promoter, totarget breast carcinoma; the hybrid myoD enhancer/SV40 promoter, whichtargets expression to rhabdomyosarcoma (RMS); the carcinoembryonicantigen (CEA) promoter, which is specific for CEA-expressing cells suchas colon cancer cells, and the hexokinase type II gene promoter, totarget non-small cell lung carcinomas. See Hart (1996), Morton & Potter(1998), Kurane et al. (1998) and Katabi et al. (1999).

Promoters that are dependent on either RNA polymerase (pol) II or pol IIare preferred promoters for gene transcription. Highly preferredpromoters for shRNA transcription are the RNA III polymerase promotersH1 and U6.

A signal sequence can be used, according to the present invention, toeffect secretion of an expression product or localization of anexpression product to a particular cellular compartment. Thus, atherapeutic polynucleotide molecule that is delivered via intact killedbacterial cells may include a signal sequence, in proper reading frame,such that the expression product of interest is secreted by an engulfingcell or its progeny, thereby to influence surrounding cells, in keepingwith the chosen treatment paradigm. Illustrative signal sequencesinclude the haemolysin C-terminal secretion sequence, described in U.S.Pat. No. 5,143,830, the BAR1 secretion sequence, disclosed in U.S. Pat.No. 5,037,743, and the signal sequence portion of the zsig32polypeptide, described in U.S. Pat. No. 6,025,197.

Reporter Elements

A nucleic acid molecule to be introduced via the approach of the presentinvention can include a reporter element. A reporter element confers onits recombinant host a readily detectable phenotype or characteristic,typically by encoding a polypeptide, not otherwise produced by the host,that can be detected, upon expression, by histological or in situanalysis, such as by in vivo imaging techniques. For example, a reporterelement delivered by an intact killed bacterial cell, according to thepresent invention, could code for a protein that produces, in theengulfing host cell, a colorimetric or fluorometric change that isdetectable by in situ analysis and that is a quantitative orsemi-quantitative function of transcriptional activation. Illustrativeof these proteins are esterases, phosphatases, proteases and otherenzymes, the activity of which generates a detectable chromophore orfluorophore.

Preferred examples are E. coli ß-galactosidase, which effects a colorchange via cleavage of an indigogenic substrate,indolyl-ß-D-galactoside, and a luciferase, which oxidizes a long-chainaldehyde (bacterial luciferase) or a heterocyclic carboxylic acid(luciferin), with the concomitant release of light. Also useful in thiscontext is a reporter element that encodes the green fluorescent protein(GFP) of the jellyfish, Aequorea victoria, as described by Prasher etal. (1995). The field of GFP-related technology is illustrated by twopublished PCT applications, WO 095/21191 (discloses a polynucleotidesequence encoding a 238 amino-acid GFP apoprotein, containing achromophore formed from amino acids 65 through 67) and WO 095/21191(discloses a modification of the cDNA for the apopeptide of A. victoriaGFP, providing a peptide having altered fluorescent properties), and bya report of Heim et al. (1994) of a mutant GFP, characterized by a4-to-6-fold improvement in excitation amplitude.

Another type of a reporter element is associated with an expressionproduct that renders the recombinant killed bacterial cell resistant toa toxin. For instance, the neo gene protects a host against toxic levelsof the antibiotic G418, while a gene encoding dihydrofolate reductaseconfers resistance to methotrexate, and the chloramphenicolacetyltransferase (CAT) gene bestows resistance to chloramphenicol.

Other genes for use as a reporter element include those that cantransform a host killed bacterial cell to express distinguishingcell-surface antigens, e.g., viral envelope proteins such as HIV gp120or herpes gD, which are readily detectable by immunoassays.

Drugs

Drugs useful in the invention may be any physiologically orpharmacologically active substance that produces a desired local orsystemic effect in animals, particularly mammals and humans. Drugs maybe inorganic or organic compounds, without limitation, includingpeptides, proteins, nucleic acids, and small molecules, any of which maybe characterized or uncharacterized. They may be in various forms, suchas unchanged molecules, molecular complexes, pharmacologicallyacceptable salts, such as hydrochloride, hydrobromide, sulfate, laurate,palmitate, phosphate, nitrite, nitrate, borate, acetate, maleate,tartrate, oleate, salicylate, and the like. For acidic drugs, salts ofmetals, amines or organic cations, for example, quaternary ammonium, canbe used. Derivatives of drugs, such as bases, esters and amides also canbe used. A drug that is water insoluble can be used in a form that is awater soluble derivative thereof, or as a base derivative thereof, whichin either instance, or by its delivery, is converted by enzymes,hydrolyzed by the body pH, or by other metabolic processes to theoriginal therapeutically active form.

Useful drugs include chemotherapeutic agents, immunosuppressive agents,cytokines, cytotoxic agents, nucleolytic compounds, radioactiveisotopes, receptors, and pro-drug activating enzymes, which may benaturally occurring or produced by synthetic or recombinant methods.

Drugs that are affected by classical multidrug resistance haveparticular utility in the invention, such as vinca alkaloids (e.g.,vinblastine and vincristine), the anthracyclines (e.g., doxorubicin anddaunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) andmicrotubule stabilizing drugs (e.g., paclitaxel). (Ambudkar et al.,1999).

In general, cancer chemotherapy agents are preferred drugs. Usefulcancer chemotherapy drugs include nitrogen mustards, nitrosorueas,ethyleneimine, alkane sulfonates, tetrazine, platinum compounds,pyrimidine analogs, purine analogs, antimetabolites, folate analogs,anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors andhormonal agents. Exemplary chemotherapy drugs are Actinomycin-D,Alkeran, Ara-C, Anastrozole, Asparaginase, BiCNU, Bicalutamide,Bleomycin, Busulfan, Capecitabine, Carboplatin, Carboplatinum,Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, CPT-11,Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan,Dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel,Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Etoposide, Floxuridine,Fludarabine, Fluorouracil, Flutamide, Fotemustine, Gemcitabine,Herceptin, Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine,Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin,Paclitaxel, Pamidronate, Pentostatin, Plicamycin, Procarbazine,Rituximab, Steroids, Streptozocin, STI-571, Streptozocin, Tamoxifen,Temozolomide, Teniposide, Tetrazine, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulphan, Trimetrexate, Vinblastine, Vincristine,Vindesine, Vinorelbine, VP-16, and Xeloda.

Useful cancer chemotherapy drugs also include alkylating agents such asThiotepa and cyclosphosphamide; alkyl sulfonates such as Busulfan,Improsulfan and Piposulfan; aziridines such as Benzodopa, Carboquone,Meturedopa, and Uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as Chlorambucil, Chlornaphazine, Cholophosphamide,Estramustine, Ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, Melphalan, Novembiehin, Phenesterine, Prednimustine,Trofosfamide, uracil mustard; nitroureas such as Cannustine,Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine;antibiotics such as Aclacinomysins, Actinomycin, Authramycin, Azaserine,Bleomycins, Cactinomycin, Calicheamicin, Carabicin, Caminomycin,Carzinophilin, Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin,6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin,Idambicin, Marcellomycin, Mitomycins, mycophenolic acid, Nogalamycin,Olivomycins, Peplomycin, Potfiromycin, Puromycin, Quelamycin,Rodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex,Zinostatin, and Zorubicin; anti-metabolites such as Methotrexate and5-fluorouracil (5-FU); folic acid analogues such as Denopterin,Methotrexate, Pteropterin, and Trimetrexate; purine analogs such asFludarabine, 6-mercaptopurine, Thiamiprine, and Thioguanine; pyrimidineanalogs such as Ancitabine, Azacitidine, 6-azauridine, Carmofur,Cytarabine, Dideoxyuridine, Doxifluridine, Enocitabine, Floxuridine, and5-FU; androgens such as Calusterone, Dromostanolone Propionate,Epitiostanol, Rnepitiostane, and Testolactone; anti-adrenals such asaminoglutethimide, Mitotane, and Trilostane; folic acid replenisher suchas frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine;Demecolcine; Diaziquone; Elformithine; elliptinium acetate; Etoglucid;gallium nitrate; hydroxyurea; Lentinan; Lonidamine; Mitoguazone;Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin;podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Razoxane;Sizofrran; Spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; Urethan; Vindesine; Dacarbazine;Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine;Arabinoside (cyclophosphamide; thiotEPa; taxoids, e.g., Paclitaxel(TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and Doxetaxel(TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); Chlorambucil;Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; platinumanalogs such as Cisplatin and Carboplatin; Vinblastine; platinum;etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine;Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin;Xeloda; Ibandronate; CPT-11 topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; Esperamicins;Capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included are anti-hormonal agentsthat act to regulate or inhibit hormone action on tumors such asanti-estrogens including for example Tamoxifen, Raloxifene, aromataseinhibiting 4(5)-imidazoles, 4 Hydroxytamoxifen, Trioxifene, Keoxifene,Onapristone, And Toremifene (Fareston); and anti-androgens such asFlutamide, Nilutamide, Bicalutamide, Leuprolide, and Goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Useful drugs also include cytokines. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-α and -ß; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-ß; platelet growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-ß; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -ß and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (GCSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumornecrosis factor such as TNF-α or TNF-ß; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

The drugs may be prodrugs, subsequently activated by aprodrug-activating enzyme that converts a prodrug like a peptidylchemotherapeutic agent to an active anti-cancer drug. See, e.g., WO88/07378; WO 81/01145; U.S. Pat. No. 4,975,278. In general, the enzymecomponent includes any enzyme capable of acting on a prodrug in such away so as to covert it into its more active, cytotoxic form.

For purposes of the invention, an intact killed bacterial cell containsa drug if it contains a nucleic acid encoding a drug. For example, aplasmid may encode a drug that is expressed inside of mammalian targetcells. This makes possible endogenous delivery of drugs, which hasadvantages over the transient nature of exogenous delivery.

Functional Nucleic Acids

“Functional nucleic acid” refers to a nucleic acid molecule that, uponintroduction into a host cell, specifically interferes with expressionof a protein. In general, functional nucleic acid molecules have thecapacity to reduce expression of a protein by directly interacting witha transcript that encodes the protein. Regulatory RNA, such as siRNA,shRNA, short RNAs (typically less than 400 bases in length), micro-RNAs(miRNAs), ribozymes and decoy RNA, and antisense nucleic acidsconstitute exemplary functional nucleic acids.

“Regulatory RNA” denotes a category inclusive of RNAs that affectexpression by RNA interference, suppression of gene expression, oranother mechanism. Accordingly, in addition to shRNA, siRNA, miRNA, andantisense ssRNA, the category of regulatory RNAs includes ribozymes anddecoy RNAs, inter alia.

Targets of Functional Nucleic Acids

Functional nucleic acids of the invention preferably target the gene ortranscript of a protein that promotes drug resistance, inhibitsapoptosis or promotes a neoplastic phenotype. Successful application offunctional nucleic acid strategies in these contexts have been achievedin the art, but without the benefits of killed bacterial cell vectors.See, e.g., Sioud (2004), Caplen (2003), Wu et al. (2003), Nieth et al.(2003), Caplen and Mousses (2003), Duxbury et al. (2004), Yague et al.(2004), Duan et al. (2004).

Proteins that contribute to drug resistance constitute preferred targetsof functional nucleic acids. The proteins may contribute to acquireddrug resistance or intrinsic drug resistance. When diseased cells, suchas tumor cells, initially respond to drugs, but become refractory onsubsequent treatment cycles, the resistant phenotype is acquired. Usefultargets involved in acquired drug resistance include ATP bindingcassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1,ABCB1, MDR-associated protein, Multidrug resistance protein 1), MDR-2and MDR-3. MRP2 (multi-drug resistance associated protein), BCR-ABL(breakpoint cluster region—Abelson protooncogene), a STI-571resistance-associated protein, lung resistance-related protein,cyclooxygenase-2, nuclear factor kappa, XRCC1 (X-ray cross-complementinggroup 1), ERCC 1 (Excision cross-complementing gene), GSTP1 (GlutathioneS-transferase), mutant β-tubulin, and growth factors such as IL-6 areadditional targets involved in acquired drug resistance. When previouslyuntreated cells fail to respond to one or more drugs, the resistantphenotype is intrinsic. An example of a protein contributing tointrinsic resistance is LRP (lung resistance-related protein).

Particularly useful targets that contribute to drug resistance includeATP binding cassette transporters such as P-glycoprotein, MDR-2, MDR-3,BCRP, APT11a and LRP.

Useful targets also include proteins that contribute to apoptosisresistance. These include Bcl-2 (B cell leukemia/lymphoma), Bcl-X_(L),A1/Bfl 1, focal adhesion kinase, Dihydrodiol dehydrogenase and p53mutant protein.

Useful targets further include oncogenic and mutant tumor suppressorproteins. Examples include ß-Catenin, PKC-α (protein kinase C), C-RAF,K-Ras (V12), DP97 Dead box RNA helicase, DNMT1 (DNA methyltransferase1), FLIP (Flice-like inhibitory protein), C-Sfc, 53BPI, Polycomb groupprotein EZH2 (Enhancer of zeste homologue), ErbB1, HPV-16 E5 and E7(human papillomavirus early 5 and early 7), Fortilin & MC11P (Myeloidcell leukemia 1 protein), DIP13α (DDC interacting protein 13a), MBD2(Methyl CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt/TCTP(Translational controlled tumor protein), SPK1 & SPK2 (Sphingosinekinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras, ErbB1, VEGF(Vascular endothelial growth factor), BAG-1 (BCL2-associated athanogene1), MRP2, BCR-ABL, STI-571 resistance-associated protein, lungresistance-related protein, cyclooxygenase-2, nuclear factor kappa,XRCC1, ERCC1, GSTP1, mutant ß-tubulin, and growth factors.

With regard to HIV infection, targets include HIV-Tat, HIV-Rev, HIV-Vif,HIV-Nef, HIV-Gag, HIV-Env, LTR, CD4, CXCR4 (chemokine receptor) and CCR5(chemokine receptor).

Because of tumor cell heterogeneity, a number of different drugresistance or apoptosis resistance pathways may be operational in targetcells. Therefore, the functional nucleic acids used in methods of theinvention may require change over time. For instance, if biopsy samplesreveal new mutations that result in acquired drug resistance, specificsiRNAs can be designed and encoded on a suitable expression plasmid,which is transformed into a killed bacterial cell-producing bacterialstrain, which is used to produce recombinant killed bacterial cells thatare administered to address the acquired drug resistance.

siRNA Molecules

Short interfering RNA (siRNA) molecules are useful for performing RNAinterference (RNAi), a post-transcriptional gene silencing mechanism.According to this invention, siRNAs refer to double-stranded RNAmolecules or single-stranded hairpin RNA molecules from about 10 toabout 30 nucleotides long, which are named for their abilityspecifically to interfere with protein expression. Preferably,double-stranded siRNA molecules are 12-28 nucleotides long, morepreferably 15-25 nucleotides long, still more preferably 19-23nucleotides long and most preferably 21-23 nucleotides long. Therefore,preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length.

The length of one strand designates the length of a double-strandedsiRNA molecule. For instance, a double-stranded siRNA that is describedas 21 ribonucleotides long (a 21-mer) could comprise two oppositestrands of RNA that anneal together for 19 contiguous base pairings. Thetwo remaining ribonucleotides on each strand would form an “overhang.”When an siRNA contains two strands of different lengths, the longer ofthe strands designates the length of the siRNA. For instance, a dsRNAcontaining one strand that is 21 nucleotides long and a second strandthat is 20 nucleotides long, constitutes a 21-mer.

Double-stranded siRNAs that comprise an overhang are desirable. Theoverhang may be at the 5′ or the 3′ end of a strand. Preferably, it isat the 3′ end of the RNA strand. The length of an overhang may vary, butpreferably is about 1 to about 5 bases, and more preferably is about 2nucleotides long. Preferably, the siRNA of the present invention willcomprise a 3′ overhang of about 2 to 4 bases. More preferably, the 3′overhang is 2 ribonucleotides long. Even more preferably, the 2ribonucleotides comprising the 3′ overhang are uridine (U).

siRNAs of the invention are designed to interact with a targetribonucleotide sequence, meaning they complement a target sequencesufficiently to hybridize to the target sequence. In one embodiment, theinvention provides an siRNA molecule comprising a ribonucleotidesequence at least 70%, 75%, 80%, 85% or 90% identical to a targetribonucleotide sequence or the complement of a target ribonucleotidesequence. Preferably, the siRNA molecule is at least 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the target ribonucleotide sequence or thecomplement of the target ribonucleotide sequence. Most preferably, ansiRNA will be 100% identical to the target nucleotide sequence or thecomplement of the ribonucleotide sequence. However, siRNA molecules withinsertions, deletions or single point mutations relative to a target mayalso be effective.

Tools to assist siRNA design are readily available to the public. Forexample, a computer-based siRNA design tool is available on the internetat www.dharmacon.com.

Relatedly, shRNAs comprise a single strand of RNA that forms a stem-loopstructure, where the stem consists of the complementary sense andantisense strands that comprise a double-stranded siRNA, and the loop isa linker of varying size. The stem structure of shRNAs generally is fromabout 10 to about 30 nucleotides long. Preferably, the stem of shRNAmolecules are 12-28 nucleotides long, more preferably 15-25 nucleotideslong, still more preferably 19-23 nucleotides long and most preferably21-23 nucleotides long. Therefore, preferred shRNA molecules comprisestems that are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27 28 or 29 nucleotides in length.

Ribozymes

Ribozymes are RNA molecules having an enzymatic activity that canrepeatedly cleave other RNA molecules in a nucleotide basesequence-specific manner. Such enzymatic RNA molecules may be targetedto virtually any RNA transcript, and efficient cleavage achieved invitro.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic polynucleotides act by first bindingto a target RNA. Such binding occurs through the target binding portionof a enzymatic polynucleotide which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic polynucleotide first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic polynucleotide hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous. Because a singleribozyme molecule is able to cleave many molecules of target RNA,effective concentrations of ribozyme can be quite low.

Useful ribozymes may comprise one of several motifs, includinghammerhead (Rossi et al. (1992)), hairpin (Hampel and Tritz, (1989),Hampel et al. (1990)), hepatitis delta virus motif (Perrotta and Been(1992), group I intron (U.S. Pat. No. 4,987,071), RNaseP RNA inassociation with an RNA guide sequence (Guerrier-Takada et al. (1983)),and Neurospora VS RNA (Saville & Collins (1990); Saville & Collins(1991); Collins & Olive (1993)). These specific motifs are not limiting,as all that is important in a ribozyme of this invention is that it hasa specific substrate binding site that is complementary to one or moretarget RNA regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule.

Ribozymes of the invention may comprise modified oligonucleotides (e.g.,for improved stability, targeting, etc.). Nucleic acid sequencesencoding the ribozymes may be under the control of a strong constitutivepromoter, such as, for example, RNA Polymerase II or RNA Polymerase IIIpromoter, so that transfected cells will produce sufficient quantitiesof the ribozyme to destroy target endogenous messages and inhibittranslation.

Antisense Oligonucleotides

Antisense oligonucleotides of the invention specifically hybridize witha nucleic acid encoding a protein, and interfere with transcription ortranslation of the protein. In one embodiment, an antisenseoligonucleotide targets DNA and interferes with its replication and/ortranscription. In another embodiment, an antisense oligonucleotidespecifically hybridizes with RNA, including pre-mRNA and mRNA. Suchantisense oligonucleotides may affect, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity that may be engaged in or facilitated by the RNA. Theoverall effect of such interference is to modulate, decrease, or inhibittarget protein expression.

“Oligonucleotide” refers to a polynucleotide comprising, for example,from about 10 nucleotides (nt) to about 1000 nt. Oligonucleotides foruse in the invention are preferably from about 10 nt to about 150 nt.The oligonucleotide may be a naturally occurring oligonucleotide or asynthetic oligonucleotide. Oligonucleotides may be modified.

“Modified oligonucleotide” and “Modified polynucleotide” refer tooligonucleotides or polynucleotides with one or more chemicalmodifications at the molecular level of the natural molecular structuresof all or any of the bases, sugar moieties, internucleoside phosphatelinkages, as well as to molecules having added substitutions or acombination of modifications at these sites. The internucleosidephosphate linkages may be phosphodiester, phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone internucleoside linkages, or3′-3′,5′-3′, or 5′-5′ linkages, and combinations of such similarlinkages. The phosphodiester linkage may be replaced with a substitutelinkage, such as phosphorothioate, methylamino, methylphosphonate,phosphoramidate, and guanidine, and the ribose subunit of thepolynucleotides may also be substituted (e.g., hexose phosphodiester;peptide nucleic acids). The modifications may be internal (single orrepeated) or at the end(s) of the oligonucleotide molecule, and mayinclude additions to the molecule of the internucleoside phosphatelinkages, such as deoxyribose and phosphate modifications which cleaveor crosslink to the opposite chains or to associated enzymes or otherproteins. The terms “modified oligonucleotides” and “modifiedpolynucleotides” also include oligonucleotides or polynucleotidescomprising modifications to the sugar moieties (e.g 3′-substitutedribonucleotides or deoxyribonucleotide monomers), any of which are boundtogether via 5′ to 3′ linkages.

There are several sites within a gene that may be utilized in designingan antisense oligonucleotide. For example, an antisense oligonucleotidemay bind the region encompassing the translation initiation codon, alsoknown as the start codon, of the open reading frame. In this regard,“start codon and “translation initiation codon” generally refer to theportion of such mRNA or gene that encompasses from at least about 25 toat least about 50 contiguous nucleotides in either direction (i.e., 5′or 3′) from a translation initiation codon.

Another site for antisense interaction to occur is the termination codonof the open reading frame. The terms “stop codon region” and“translation termination codon region” refer generally to a portion ofsuch a mRNA or gene that encompasses from at least about 25 to at leastabout 50 contiguous nucleotides in either direction form a translationtermination codon.

The open reading frame or coding region also may be targetedeffectively. The open reading frame is generally understood to refer tothe region between the translation initiation codon and the translationtermination codon. Another target region is the 5′ untranslated region,which is the portion of a mRNA in the 5′ direction from the translationinitiation codon. It includes the nucleotides between the 5′ cap siteand the translation initiation codon of a mRNA or correspondingnucleotides on the gene.

Similarly, the 3′ untranslated region may be used as a target forantisense oligonucleotides. The 3′ untranslated region is that portionof the mRNA in the 3′ direction from the translation termination codon,and thus includes the nucleotides between the translation terminationcodon and the 3′ end of a mRNA or corresponding nucleotides of the gene.

An antisense oligonucleotide may also target the 5′ cap region of anmRNA. The 5′ cap comprises an N7-methylated guanosine residue joined tothe 5′-most residue of the mRNA via 5′-5′ triphosphate linkage. The 5′cap region is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more intron regions, which are excised from a transcriptbefore it is translated. The remaining (and therefore translated) exonregions are spliced together to form a continuous mRNA sequence. mRNAsplice sites, i.e., intron-exon junctions, represent possible targetregions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Moreover,aberrant fusion junctions due to rearrangements or deletions are alsopossible targets for antisense oligonucleotides.

With these various target sites in mind, antisense oligonucleotides thatare sufficiently complementary to the target polynucleotides must bechosen. “Complementary” refers to the topological compatibility ormatching together of the interacting surfaces of two molecules. Theremust be a sufficient degree of complementarity or precise pairing suchthat stable and specific binding occurs between the oligonucleotide andthe polynucleotide target. Importantly, the sequence of an antisenseoligonucleotide need not be 100% complementary to that of its targetpolynucleotide to be specifically hybridizable. An antisenseoligonucleotide is specifically hybridizable when binding of theantisense oligonucleotide to the target polynucleotide interferes withthe normal function of the target polynucleotide to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, and in the case of in vitro assays, underconditions in which the assays are performed.

The antisense oligonucleotides may be at least about 8 nt to at leastabout 50 nt in length. In one embodiment, the antisense oligonucleotidesmay be about 12 to about 30 nt in length.

The antisense oligonucleotides used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Nucleic Acids Encoding Functional Nucleic Acids

For purposes of the invention, an intact killed bacterial cell containsa functional nucleic acid if it contains a nucleic acid encoding afunctional nucleic acid. For example, a plasmid may encode a functionalnucleic acid that is expressed inside of mammalian target cells. Thismakes possible endogenous delivery of functional nucleic acids, whichhas advantages over the transient nature of exogenous delivery.

Thus, recombinant intact killed bacterial cells may carry plasmid DNAencoding one or more siRNA sequences aimed at silencing drug resistanceor apoptosis resistance genes. Using killed bacterial cells that encodemultiple functional nucleic acids, it is possible to treat cells thatexpress multiple drug resistance mechanisms. Different siRNA sequencescan be expressed individually from different promoters. For example,siRNA targeting Pgp mRNA can be expressed from the U6 promoter and siRNAtargeting Bcl-2 mRNA can be expressed from the 111 promoter. Thesemultiple expression cassettes preferably are carried on a singleplasmid, but may also be on different plasmids. Different siRNAsequences also can be expressed from a single promoter, where therecombinant plasmid carries an expression cassette comprised of multiplesiRNA-encoding sequences, which are linked together via non-codingpolynucleotide sequences. A single gene transcription terminator can beplaced downstream of the complete expression cassette.

In one strategy, a plasmid encodes the sense and antisense strands of ansiRNA as two independent transcripts that, after expression within atarget cell, hybridize to form functional siRNA duplexes. In a secondpreferred strategy, a plasmid encodes one or more siRNAs that each areexpressed as a single transcript that forms a short hairpin RNAstem-loop structure. The hairpin structure may be processed by a Dicerenzyme into functional siRNA.

Pharmaceutically Acceptable Carriers

“Pharmaceutically acceptable” refers to physiological compatibility. Apharmaceutically acceptable carrier or excipient does not abrogatebiological activity of the composition being administered, is chemicallyinert and is not toxic to the organism in which it is administered.

Endotoxin

“Endotoxin” refers to free lipopolysaccharide (LPS). Accordingly, acomposition that is “free of endotoxin” lacks LPS that is unassociatedwith a bacterial cell membrane. A composition that is “essentially freeof endotoxin” lacks a sufficient quantity or concentration of LPS tocause toxicity in a mammal, such as a human, Endotoxin/LPS that isunassociated with a bacterial cell membrane also is referred to as “freeendotoxin.”

Endotoxin can be eliminated from a composition via filtration through a0.2 μm filter. Free endotoxin and endotoxin micelles are smaller than0.2 μm and hence are readily filtered from a composition that retainskilled bacterial cells, which are larger than 0.2 μm. Additionally,anti-lipid A monoclonal antibodies can be used to bind to freeendotoxin. The anti-lipid A monoclonal antibodies can be bound to asolid support such as an affinity chromatography column or magneticbeads via their Fc component, leaving the lipid A-binding Fab fragmentsfree to bind to free LPS.

Bispecific Ligands

Compositions of the invention also may comprise one or more bispecificligands. Ligands useful in the invention include any agent that binds toa surface component on a target cell and to a surface component on akilled bacterial cell. Preferably, the surface component on a targetcell is a receptor, especially a receptor capable of mediatingendocytosis. The ligands may comprise a polypeptide and/or carbohydratecomponent. Antibodies are preferred ligands. For example, a bispecificantibody that carries dual specificities for a surface component onbacterially derived intact killed bacterial cells and for a surfacecomponent on target mammalian cells, can be used efficiently to targetthe killed bacterial cells to the target mammalian cells in vitro and invivo. The category of useful ligands also includes receptors, enzymes,binding peptides, fusion/chimeric proteins and small molecules.

The selection of a particular ligand is made on two primary criteria:(i) specific binding to one or more domains on the surface of intactkilled bacterial cells and (ii) specific binding to one or more domainson the surface of the target cells. Thus, ligands preferably have afirst arm that carries specificity for a bacterially derived intactkilled bacterial cell surface structure and a second arm that carriesspecificity for a mammalian cell surface structure. Each of the firstand second arms may be multivalent. Preferably, each arm ismonospecific, even if multivalent.

For binding to bacterially derived killed bacterial cells, it isdesirable for one arm of the ligand to be specific for theO-polysaccharide component of a lipopolysaccharide found on the parentbacterial cell. Other killed bacterial cell surface structures that canbe exploited for ligand binding include cell surface-exposedpolypeptides and carbohydrates on outer membranes, such as pilli,fimbrae, outer-membrane protein and flagella cell surface exposedpeptide segments.

For binding to target cells, one arm of the ligand is specific for asurface component of a mammalian cell. Such components include cellsurface proteins, peptides and carbohydrates, whether characterized oruncharacterized. Cell surface receptors, especially those capable ofactivating receptor-mediated endocytosis, are desirable cell surfacecomponents for targeting. Such receptors, if over-expressed on thetarget cell surface, confer additional selectivity for targeting thecells to be treated, thereby reducing the possibility for delivery tonon-target cells.

By way of example, one may target tumor cells, metastatic cells,vasculature cells, such as endothelial cells and smooth muscle cells,lung cells, kidney cells, blood cells, bone marrow cells, brain cells,liver cells, and so forth, or precursors of any selected cell byselecting a ligand that specifically binds a cell surface receptor motifon the desired cells. Examples of cell surface receptors includecarcinoembryonic antigen (CEA), which is overexpressed in most colon,rectum, breast, lung, pancreas and gastrointestinal tract carcinomas(Marshall, 2003); heregulin receptors (HER-2, neu or c-erbB-2), which isfrequently overexpressed in breast, ovarian, colon, lung, prostate andcervical cancers (Hung et al., 2000); epidermal growth factor receptor(EGFR), which is highly expressed in a range of solid tumors includingthose of the breast, head and neck, non-small cell lung and prostate(Salomon et al., 1995); asialoglycoprotein receptor (Stockert, 1995);transferrin receptor (Singh, 1999); serpin enzyme complex receptor,which is expressed on hepatocytes (Ziady et al., 1997); fibroblastgrowth factor receptor (EGFR), which is overexpressed on pancreaticductal adenocarcinoma cells (Kleeff et al., 2002); vascular endothelialgrowth factor receptor (VEGFR), for anti-angiogenesis gene therapy(Becker et al., 2002 and Hoshida et al., 2002); folate receptor, whichis selectively overexpressed in 90% of nonmucinous ovarian carcinomas(Gosselin and Lee, 2002); cell surface glycocalyx (Batra et al. 1994);carbohydrate receptors (Thurnher et al., 1994); and polymericimmunoglobulin receptor, which is useful for gene delivery torespiratory epithelial cells and attractive for treatment of lungdiseases such as Cystic Fibrosis (Kaetzel et al., 1997).

Preferred ligands comprise antibodies and/or antibody derivatives. Asused herein, the term “antibody” encompasses an immunoglobulin moleculeobtained by in vitro or in vivo generation of an immunogenic response.The term “antibody” includes polyclonal, monospecific and monoclonalantibodies, as well as antibody derivatives, such as single-chainantibody fragments (scFv). Antibodies and antibody derivatives useful inthe present invention also may be obtained by recombinant DNAtechniques.

Wild type antibodies have four polypeptide chains, two identical heavychains and two identical light chains. Both types of polypeptide chainshave constant regions, which do not vary or vary minimally amongantibodies of the same class, and variable regions. Variable regions areunique to a particular antibody and comprise an antigen binding domainthat recognizes a specific epitope. The regions of the antigen bindingdomain that are most directly involved in antibody binding are“complementarity-determining regions” (CDRs).

The term “antibody” also encompasses derivatives of antibodies, such asantibody fragments that retain the ability to specifically bind toantigens. Such antibody fragments include Fab fragments (a fragment thatcontains the antigen-binding domain and comprises a light chain and partof a heavy chain bridged by a disulfide bond), Fab′ (an antibodyfragment containing a single antigen-binding domain comprising a Fab andan additional portion of the heavy chain through the hinge region,F(ab′)2 (two Fab′ molecules joined by interchain disulfide bonds in thehinge regions of the heavy chains), a bispecific Fab (a Fab moleculehaving two antigen binding domains, each of which may be directed to adifferent epitope), and an scFv (the variable, antigen-bindingdeterminative region of a single light and heavy chain of an antibodylinked together by a chain of amino acids).

When antibodies, including antibody fragments, constitute part or all ofthe ligands, they preferably are of human origin or are modified to besuitable for use in humans. So-called “humanized antibodies” are wellknown in the art. See, e.g., Osbourn et al., 2003. They have beenmodified by genetic manipulation and/or in vitro treatment to reducetheir antigenicity in a human. Methods for humanizing antibodies aredescribed, e.g., in U.S. Pat. Nos. 6,639,055, 5,585,089, and 5,530,101.In the simplest case, humanized antibodies are formed by grafting theantigen-binding loops, known as complementarity-determining regions(CDRs), from a mouse mAb into a human IgG. See Jones et al., 1986;Riechmann et al., 1988; and Verhoeyen et al., 1988. The generation ofhigh-affinity humanized antibodies, however, generally requires thetransfer of one or more additional residues from the so-called frameworkregions (FRs) of the mouse parent mAb. Several variants of thehumanization technology also have been developed. See Vaughan et al.,1998.

Human antibodies, rather than “humanized antibodies,” also may beemployed in the invention. They have high affinity for their respectiveantigens and are routinely obtained from very large, single-chainvariable fragments (scFvs) or Fab phage display libraries. See Griffithset al., 1994; Vaughan et al., 1996; Sheets et al., 1998; de Haard etal., 1999; and Knappik et al., 2000.

Useful ligands also include bispecific single chain antibodies, whichtypically are recombinant polypeptides consisting of a variable lightchain portion covalently attached through a linker molecule to acorresponding variable heavy chain portion. See U.S. Pat. Nos.5,455,030, 5,260,203, and 4,496,778. Bispecific antibodies also can bemade by other methods. For example, chemical heteroconjugates can becreated by chemically linking intact antibodies or antibody fragments ofdifferent specificities. See Karpovsky et al., 1984. However, suchheteroconjugates are difficult to make in a reproducible manner and areat least twice as large as normal monoclonal antibodies. Bispecificantibodies also can be created by disulfide exchange, which involvesenzymatic cleavage and reassociation of the antibody fragments. SeeGlennie et al., 1987.

Because Fab and scFv fragments are monovalent they often have lowaffinity for target structures. Therefore, preferred ligands made fromthese components are engineered into dimeric, trimeric or tetramericconjugates to increase functional affinity. See Tomlinson and Holliger,2000; Carter, 2001; Hudson and Souriau, 2001; and Todorovska et al.,2001. Such conjugate structures may be created by chemical and/orgenetic cross-links.

Bispecific ligands of the invention preferably are monospecific at eachend, i.e., specific for a single component on killed bacterial cells atone end and specific for a single component on target cells at the otherend. The ligands may be multivalent at one or both ends, for example, inthe form of so-called diabodies, triabodies and tetrabodies. See Hudsonand Souriau, 2003. A diabody is a bivalent dimer formed by anon-covalent association of two scFvs, which yields two Fv bindingsites. Likewise, a triabody results from the formation of a trivalenttrimer of three scFvs, yielding three binding sites, and a tetrabodyresults from the formation of a tetravalent tetramer of four scFvs,yielding four binding sites.

Several humanized, human, and mouse monoclonal antibodies and fragmentsthereof that have specificity for receptors on mammalian cells have beenapproved for human therapeutic use, and the list is growing rapidly. SeeHudson and Souriau, 2003. An example of such an antibody that can beused to form one arm of a bispecific ligand has specificity for HER2:Herceptin™; Trastuzumab.

Antibody variable regions also can be fused to a broad range of proteindomains. Fusion to human immunoglobulin domains such as IgG1 CH3 bothadds mass and promotes dimerization. See Hu et al., 1996. Fusion tohuman Ig hinge-Fc regions can add effector functions. Also, fusion toheterologous protein domains from multimeric proteins promotesmultimerization. For example, fusion of a short scFv to shortamphipathic helices has been used to produce miniantibodies. See Packand Pluckthun, 1992. Domains from proteins that form heterodimers, suchas fos/jun, can be used to produce bispecific molecules (Kostelny etal., 1992) and, alternately, homodimerization domains can be engineeredto form heterodimers by engineering strategies such as “knobs intoholes” (Ridgway et al., 1996). Finally, fusion protein partners can beselected that provide both multimerization as well as an additionalfunction, e.g. streptavidin. See Dubel et al., 1995.

Additional Compositions

In one embodiment, the composition comprises a killed bacterial cellthat contains a functional nucleic acid molecule and a drug. Thefunctional nucleic acid molecule may be one that targets the transcriptof a protein that contributes to drug resistance. Preferably, thefunctional nucleic acid molecule targets the transcript of a proteinthat contributes to resistance against the same drug in the composition.The drug may be contained within a killed bacterial cell, even the samekilled bacterial cell as the functional nucleic acid molecule, but neednot be so contained.

Delivery Methods to Phagocytosis- or Endocytosis-Competent Cells

In another aspect, the invention provides for delivery by means ofbringing bacterially derived killed bacterial cells into contact withmammalian cells that are phagocytosis- or endocytosis-competent. Suchmammalian cells, which are capable of engulfing parent bacterial cellsin the manner of intracellular bacterial pathogens, likewise engulf thekilled bacterial cells, which release their payload into the cytoplasmof the mammalian cells. This delivery approach can be effected withoutthe use of targeting ligands.

A variety of mechanisms may be involved in the engulfing of killedbacterial cells by a given type of cell, and the present invention isnot dependent on any particular mechanism in this regard. For example,phagocytosis is a well-documented process in which macrophages and otherphagocyte cells, such as neutrophils, ingest particles by extendingpseudopodia over the particle surface until the particle is totallyenveloped. Although described as “non-specific” phagocytosis, theinvolvement of specific receptors in the process has been demonstrated.See Wright & Jong (1986): Speert et al. (1988).

Thus, one form of phagocytosis involves interaction between surfaceligands and ligand-receptors located at the membranes of the pseudopodia(Shaw and Griffin, 1981). This attachment step, mediated by the specificreceptors, is thought to be dependent on bacterial surface adhesins.With respect to less virulent bacteria, such as non-enterotoxigenic E.coli, phagocytosis also may occur in the absence of surface ligands forphagocyte receptors. See Pikaar et al. (1995), for instance. Thus, thepresent invention encompasses but is not limited to the use of killedbacterial cells that either possess or lack surface adhesins, in keepingwith the nature of their parent bacterial cells, and are engulfed byphagocytes (i.e., “phagocytosis-competent” host cells), of whichneutrophils and macrophages are the primary types in mammals.

Another engulfing process is endocytosis, by which intracellularpathogens exemplified by species of Salmonella, Escherichia, Shigella,Helicobacter, Pseudomonas and Lactobacilli gain entry to mammalianepithelial cells and replicate there. Two basic mechanisms in thisregard are Clathrin-dependent receptor-mediated endocytosis, also knownas “coated pit endocytosis” (Riezman, 1993), and Clathrin-independentendocytosis (Sandvig & Deurs, 1994). Either or both may be involved whenan engulfing-competent cell that acts by endocytosis (i.e., an“endocytosis-competent” host cell) engulfs killed bacterial cells inaccordance with the invention. Representative endocytosis-competentcells are breast epithelial cells, enterocytes in the gastrointestinaltract, stomach epithelial cells, lung epithelial cells, and urinarytract and bladder epithelial cells.

When effecting delivery to an engulfing-competent mammalian cell withoutthe use of a targeting ligand, the nature of the applicationcontemplated will influence the choice of bacterial source for thekilled bacterial cells employed. For example, Salmonella, Escherichiaand Shigella species carry adhesins that are recognized byendocytosis-mediating receptors on enterocytes in the gastrointestinaltract, and may be suitable to deliver a drug that is effective for coloncancer cells. Similarly, killed bacterial cells derived fromHelicobacter pylori, carrying adhesins specific for stomach epithelialcells, could be suited for delivery aimed at stomach cancer cells.Inhalation or insufflation may be ideal for administering intact killedbacterial cells derived from a Pseudomonas species that carry adhesinsrecognized by receptors on lung epithelial cells. Killed bacterial cellsderived from Lactobacilli bacteria, which carry adhesins specific forurinary tract and bladder epithelial cells, could be well-suited forintraurethral delivery of a drug to a urinary tract or a bladder cancer.

In one embodiment, the delivery method is a therapeutic nucleic aciddelivery method that comprises bringing killed bacterial cells thatcontain a plasmid comprised of a nucleic acid sequence into contact withmammalian cells that are phagocytosis- or endocytosis-competent, suchthat the killed bacterial cells are engulfed by the mammalian cells. Theplasmid preferably encodes a therapeutic expression product. After thekilled bacterial cells are brought into contact with the mammaliancells, the latter cells produce an expression product of the therapeuticnucleic acid sequence. The therapeutic nucleic acid delivery method maybe performed in vitro or in vivo.

In another embodiment, the delivery method is a drug delivery methodthat comprises bringing killed bacterial cells that contain a drug intocontact with mammalian cells that are phagocytosis- orendocytosis-competent, such that the killed bacterial cells are engulfedby the mammalian cells. The drug is then released into the cytoplasm ofthe mammalian cells. Alternatively, the killed bacterial cells maycontain a plasmid that encodes a drug, in which case the plasmidoptionally comprises a regulatory element and/or a reporter element. Thedrug delivery method may be performed in vitro or in vivo.

In another embodiment, the delivery method is a functional nucleic aciddelivery method that comprises bringing a killed bacterial cell thatcontains either a functional nucleic acid molecule or a plasmid thatencodes a functional nucleic acid molecule into contact with mammaliancells that are phagocytosis- or endocytosis-competent, such that thekilled bacterial cells are engulfed by the mammalian cells. Thefunctional nucleic acid or plasmid is then released into the mammaliancell. In the case that the killed bacterial cell contains a plasmidencoding a functional nucleic acid molecule, the plasmid optionallycomprises a regulatory element and/or a reporter element and themammalian cell preferably expresses the functional nucleic acid. Thefunctional nucleic acid delivery method may be performed in vitro or invivo.

Thus, in one aspect a method of delivering functional nucleic acidinvolves the use of killed bacterial cells that comprise plasmid-freefunctional nucleic acid. In this regard, functional nucleic acids arepackaged directly into killed bacterial cells by passing through thebacterial cell's intact membrane without using plasmid-based expressionconstructs or the expression machinery of a host cell. In oneembodiment, therefore, a method of delivering functional nucleic acidcomprises (a) providing a plurality of intact killed bacterial cells ina pharmaceutically acceptable carrier, each bacterial cell of theplurality encompassing plasmid-free functional nucleic acid, and (b)bringing the killed bacterial cells of the plurality into contact withmammalian cells such that the mammalian cells engulf killed bacterialcells of the plurality, whereby the functional nucleic acid is releasedinto the cytoplasm of the target cells.

The qualifier “plasmid-free” connotes the absence of a construct, suchas a plasmid or viral vector, for in situ expression of a regulatoryRNA.

Directing Killed Bacterial Cells to Specific Mammalian Cells

In another aspect, the invention provides for targeted delivery mediatedthat employs a bispecific ligand. The ligand brings a killed bacterialcell into contact with a target mammalian cell, such that the mammaliancell engulfs the killed bacterial cell, including the killed bacterialcell's payload.

In one embodiment, the targeted delivery method is a therapeutic nucleicacid delivery method that comprises bringing bispecific ligands intocontact with killed bacterial cells that contain a therapeutic nucleicacid sequence and non-phagocytic mammalian cells. The bispecific ligandscause the killed bacterial cells to bind to the mammalian cells, and thekilled bacterial cells become engulfed by the mammalian cells. Themammalian cells may then produce an expression product of thetherapeutic nucleic acid.

The efficiency of nucleic acid delivery relates to the copy number ofplasmid DNA that the killed bacterial cells carry. It is well known thata bottleneck of nucleic acid delivery is that >99% of the internalizedDNA is degraded in the endosome or lysosome, without reaching thecytoplasm of the target cell. As a non-living particle, killed bacterialcells are expected to lack functions destabilizing or disrupting theendo-lysosomal membrane of target cells and are unlikely to possesssophisticated mechanisms for allowing internalized DNA to escape theendo-lysosomal membrane. Pursuant to the present invention, therefore,killed bacterial cells carrying at least 70 to 100 copies of plasmid DNAare preferred. The inventors have used such killed bacterial cells forsuccessful nucleic acid delivery. The successful result suggests thateven if most of the plasmid DNA is degraded in the endo-lysosomalvacuole, it is possible to overwhelm the system and to have some DNA toescape intact into the mammalian cell cytoplasm.

In another embodiment, the targeted delivery method is a drug deliverymethod that comprises bringing bispecific ligands into contact withkilled bacterial cells that contain a drug molecule and non-phagocyticmammalian cells. The bispecific ligands cause the killed bacterial cellsto bind to the mammalian cells, and the killed bacterial cells becomeengulfed by the mammalian cells. The drug molecule then is released intothe cytoplasm of the mammalian cells.

The inventors have discovered that a significant concentration of thedrug carried by bispecific ligand-targeted killed bacterial cells alsoescapes the endo-lysosomal membrane and enters the mammalian cellcytoplasm. Moreover, the killed bacterial cells are highly versatile intheir capacity to package a range of different drugs, e.g., hydrophilic,hydrophobic, and amphipathic, such as doxorubicin, paclitaxel,cisplatin, carboplatin, 5-fluorouracil, irinotecan. All these drugs arereadily packaged in killed bacterial cells in therapeuticallysignificant concentrations.

In another embodiment, the targeted delivery method is a functionalnucleic acid delivery method that comprises bringing bispecific ligandsinto contact with (a) killed bacterial cells that contain a functionalnucleic acid molecule or a plasmid comprised of a segment that encodes afunctional nucleic acid molecule and (b) target mammalian cells. Thebispecific ligands cause the killed bacterial cells to bind to themammalian cells, and the killed bacterial cells become engulfed by themammalian cells. Following engulfment of the killed bacterial cell, thefunctional nucleic acid molecule is released into the cytoplasm of thetarget cell or expressed by the target cell.

These targeted delivery methods may be performed in vivo or in vitro, orboth in vivo and in vitro. Contact between bispecific ligand, killedbacterial cell and mammalian cell may occur in a number of differentways. For in vivo delivery, it is preferable to administer a killedbacterial cell that already has the bispecific ligand attached to it.Thus, killed bacterial cell, bispecific ligand and target cell all arebrought into contact when the bispecific ligand-targeted killedbacterial cell reaches the target cell in vivo. Alternatively,bispecific ligand and killed bacterial cell can be separatelyadministered in vivo.

Contact between the bispecific ligands, killed bacterial cells andmammalian cells also may occur during one or more incubations in vitro.In one embodiment, the three elements are incubated together all atonce. Alternatively, step-wise incubations may be performed. In oneexample of a step-wise approach, killed bacterial cells and bi-specificligands are first incubated together to form bispecific ligand-targetedkilled bacterial cells, which are then incubated with target cells. Inanother example, bispecific ligands are first incubated with targetcells, followed by an incubation with killed bacterial cells. Acombination of one or more in vitro incubations and in vivoadministrations also may bring bispecific ligands, killed bacterialcells and mammalian target cells into contact.

The inventors found that the targeted delivery approach is broadlyapplicable to a range of mammalian cells, including cells that normallyare refractory to specific adhesion and endocytosis of killed bacterialcells. For example, bispecific antibody ligands withanti-O-polysaccharide specificity on one arm and anti-HER2 receptor,anti-EGF receptor or anti-androgen receptor specificity on the other armefficiently bind killed bacterial cells to the respective receptors on arange of target non-phagocytic cells. These cells include lung, ovarian,brain, breast, prostate and skin cancer cells. Moreover, the efficientbinding precedes rapid endocytosis of the killed bacterial cells by eachof the non-phagocytic cells.

Target cells of the invention include any cell into which a therapeuticnucleic acid, drug or functional nucleic acid is to be introduced.Desirable target cells are characterized by expression of a cell surfacereceptor that, upon binding of a ligand, facilitates endocytosis.Preferred target cells are non-phagocytic, meaning that the cells arenot professional phagocytes, such as macrophages, dendritic cells andNatural Killer (NK) cells. Preferred target cells also are mammalian.

Delivery methods of the invention may be employed for the purpose oftreating disease conditions. The terms “treatment,” “treating,” “treat,”and the like refer to obtaining a desired pharmacological and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” covers any treatment of a disease in a mammal, particularlya human, and includes: (a) preventing the disease or symptom fromoccurring in a subject which may be predisposed to the disease orsymptom but has not yet been diagnosed as having it; (b) inhibiting thedisease symptom, i.e., arresting its development; or (c) relieving thedisease symptom, i.e., causing regression of the disease or symptom.

Use of Functional Nucleic Acids to Overcome Drug Resistance and TreatDisease

In another aspect, the invention provides a method of overcoming drugresistance and treating a disease, such as cancer or AIDS, in a subjectthrough the use of functional nucleic acids. The method comprises (a)providing an intact killed bacterial cell that contains a functionalnucleic acid molecule or a plasmid comprising a segment that encodes afunctional nucleic acid molecule, where the functional nucleic acidmolecule targets the gene or transcript of a protein that promotes drugresistance, (b) bringing the killed bacterial cell into contact with atarget mammalian cell, such that the mammalian cell engulfs the killedbacterial cell, and (c) delivering a drug to the target mammalian cell.Preferably, step (c) is performed after steps (a) and (b), to allow thefunctional nucleic acid to diminish resistance to the drug prior to thedrug's administration. Delivery of the drug and introduction of thefunctional nucleic acid can occur consecutively, in any order, orsimultaneously.

Drugs may be delivered by any conventional means. For example, drugs maybe delivered orally, parenterally (including subcutaneously,intravenously, intramuscularly, intraperitoneally, and by infusion),topically, transdermally or by inhalation. The appropriate mode ofdelivery and dosage of each drug is easily ascertainable by thoseskilled in the medical arts.

Although drug delivery may occur via conventional means, delivery viakilled bacterial cells is preferred. In this regard, the inventors havediscovered that the same mammalian cells can be successfullyre-transfected by targeted intact killed bacterial cells that arepackaged with different payloads. For example, siRNA-encodingplasmid-packaged killed bacterial cells can transfect a mammalian cell,after which drug-packaged killed bacterial cells can deliver drug to thesame mammalian cell. This discovery was a surprise, and indicates thatthe intracellular processes associated with killed bacterial cellbreakdown, endosomal release of a payload and escape of the payload tointracellular targets remains fully functional after the first round oftransfection and payload delivery.

The drug may be packaged in a separate killed bacterial cell from thefunctional nucleic acid or plasmid encoding the functional nucleic acid.Alternatively, the drug may be packaged in the same killed bacterialcell as the functional nucleic acid molecule or plasmid encoding thefunctional nucleic acid molecule. Certain drugs may interact withnucleic acids and preclude co-packaging of drug and nucleic acid in thesame killed bacterial cell. For example, Doxorubicin is known tointeract with DNA.

Packaging Functional Nucleic Acid into Killed Bacterial Cells

Functional nucleic acid can be packaged directly into intact killedbacterial cells. The process bypasses the previously required steps offor example, cloning nucleic acids encoding regulatory RNA intoexpression plasmids, transforming minicell-producing parent bacteriawith the plasmids and generating recombinant minicells. Instead,plasmid-free functional nucleic acid can be packaged directly intokilled bacterial cells by co-incubating a plurality of intact killedbacterial cells with functional nucleic acid in a buffer.

In some embodiments, the co-incubation may involve gentle shaking, whilein others the co-incubation is static. A co-incubation of about one houris sufficient, but shorter periods, such as about half an hour, also maybe effective. In one embodiment, the buffer comprises buffered saline,for example a 1× phosphate buffer solution. The buffered saline can bein gelatin form. In another embodiment, the co-incubation is conductedat a temperature of about 4° C. to about 37° C.; about 20° C. to about30° C.; about 25° C.; or about 37° C. In other aspects, theco-incubation can comprise about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or 10¹³killed bacterial cells. Specific parameters of temperature, time,buffer, minicell concentration, etc. can be optimized for a particularcombination of conditions.

Loading Killed Bacteria with Drugs

Preferably, killed bacterial cells of the invention contain a sufficientquantity of drug to exert the drug's physiological or pharmacologicaleffect on a target cell. Also preferably, drugs contained within thekilled bacterial cells are heterologous, or foreign, to the killedbacterial cells, meaning that the killed bacterial cells' parentbacterial cells do not normally produce the drug.

Both hydrophilic and hydrophobic drugs can be packaged in killedbacterial cells by creating a concentration gradient of the drug betweenan extracellular medium containing killed bacterial cells and the killedbacterial cell cytoplasm. When the extracellular medium contains ahigher drug concentration than the killed bacterial cell cytoplasm, thedrug naturally moves down this concentration gradient, into the killedbacterial cell cytoplasm. When the concentration gradient is reversed,however, the drug does not move out of the killed bacterial cells.

To load killed bacterial cells with drugs that normally are not watersoluble, the drugs initially can be dissolved in an appropriate solvent.For example, Paclitaxel can be dissolved in a 1:1 blend of ethanol andcremophore EL (polyethoxylated castor oil), followed by a dilution inPBS to achieve a solution of Paclitaxel that is partly diluted inaqueous media and carries minimal amounts of the organic solvent toensure that the drug remains in solution. Killed bacterial cells can beincubated in this final medium for drug loading. Thus, the inventorsdiscovered that even hydrophobic drugs can diffuse into the cytoplasm ofkilled bacterial cells to achieve a high and therapeutically significantcytoplasmic drug load. This is unexpected because the killed bacterialcell membrane is composed of a hydrophobic phospholipid which would beexpected to prevent diffusion of hydrophobic molecules into thecytoplasm.

Another method of loading killed bacterial cells with a drug involvesculturing a recombinant parent bacterial cell under conditions whereinthe parent bacterial cell transcribes and translates a nucleic acidencoding the drug, such that the drug is released into the cytoplasm ofthe parent bacterial cell. For example, a gene cluster encoding thecellular biosynthetic pathway for a desired drug can be cloned andtransferred into a parent bacterial strain that is capable of producingkilled bacterial cells. Genetic transcription and translation of thegene cluster results in biosynthesis of the drug within the cytoplasm ofthe parent bacterial cells, filling the bacterial cytoplasm with thedrug. When the parent bacterial cell divides and forms progeny killedbacterial cells, the killed bacterial cells also contain the drug intheir cytoplasm. The pre-packaged killed bacterial cells can be purifiedby any of the killed bacterial cell purification processes known in theart and described above.

Similarly, another method of loading killed bacterial cells with a druginvolves culturing a recombinant killed bacterial cell that contains anexpression plasmid encoding the drug under conditions such that the geneencoding the drug is transcribed and translated within the killedbacterial cell.

Purity of Compositions

Killed bacterial cells of the invention are substantially free fromcontaminating parent bacterial cells, i.e., live bacterial cells. Thus,killed bacterial cell-containing compositions of the inventionpreferably contain fewer than about 1 contaminating parent bacterialcell per 10⁷ killed bacterial cells, more preferably contain fewer thanabout 1 contaminating parent bacterial cell per 10⁸ killed bacterialcells, even more preferably contain fewer than about 1 contaminatingparent bacterial cell per 10⁹ killed bacterial cells, still morepreferably contain fewer than about 1 contaminating parent bacterialcell per 10¹⁰ killed bacterial cells and most preferably contain fewerthan about 1 contaminating parent bacterial cell per 10¹¹ killedbacterial cells.

A composition consisting essentially of killed bacterial cells and,optionally therapeutic nucleic acids, drugs, functional nucleic acidsand bispecific ligands, of the present invention (that is, a formulationthat includes such killed bacterial cells with other constituents thatdo not interfere unduly with the delivering quality of the composition)can be formulated in conventional manner, using one or morepharmaceutically acceptable carriers or excipients.

Bacterial cells in culture can be killed using a number of differentprocedures including (a) treatment with an antibiotic to which thebacterial strain is sensitive, (b) treatment with heat that is below thelevel at which protein coagulation occurs, and (c) treatment withsolvents like ethanol at a concentration that does not result in loss ofbacterial cell integrity and closure of protein channels in thebacterial membrane. The bacterial cell killing process is well known inthe art of manufacture of killed bacterial vaccines. Preferably, theprocess of bacterial cell killing does not involve extensivedenaturation of the spatial configuration of the molecules; that is, theprocess preferably preserves the three-dimensional structure ofmacromolecules from the bacteria cells, such as proteins,polysaccharides and lipids. Other processes that may be used forobtaining the killed bacterial preparation as defined above are known tothose of ordinary skill in the art.

The absence of membrane denaturation in a killed bacterial preparationcan be verified by any method well-known in the art. For example,plasmid DNA can be extracted from recombinant killed bacterial cells andcan be sequenced to ascertain integrity of the recombinant DNA. Plasmidcontent can be determined by Real-time PCR and compared to plasmidcontent in the same number of live recombinant bacterial cells. Ifmembrane integrity was not preserved in the killing process, thenplasmid loss would be expected to occur. Additionally, if a killingprocess damaged recombinant plasmid, then DNA sequence aberrations wouldbe observed. A test can also be conducted where the same number of liveand killed bacterial cells are checked for the ability to package achemotherapeutic drug.

Impurities such as media, buffers, cellular debris, membrane blebs, freenucleic acids and free endotoxin can be eliminated from a killedbacterial preparation by filtration, such as filtration through 0.2 μmcross-flow filtration. A filter pore size of about 0.2 μm is preferredbecause contaminants generally are smaller than 0.2 μm. Thus, using sucha filter pore size allows contaminants to be filtered out, and intactkilled bacterial to be retained. The filtration may be dead-endfiltration or cross-flow filtration. Cross-flow filtration has theadvantage of less filter clogging. Also, it is preferable to performbuffer exchange washing steps, which also can employ a filter pore sizeof about 0.2 μm.

Administration Routes and Form of Compositions

Compositions of the invention can be administered via various routes andto various sites in a mammalian body, to achieve the therapeuticeffect(s) desired, either locally or systemically. Delivery may beaccomplished, for example, by oral administration, by application of theformulation to a body cavity, by inhalation or insufflation, or byparenteral, intramuscular, intravenous, intraportal, intrahepatic,peritoneal, subcutaneous, intratumoral, or intradermal administration.The mode and site of administration is dependent on the location of thetarget cells. For example, cystic-fibrotic cells may be efficientlytargeted by inhaled delivery of the targeted killed bacterial cells.Similarly, tumor metastasis may be more efficiently treated viaintravenous delivery of targeted killed bacterial cells. Primary ovariancancer may be treated via intraperitoneal delivery of targeted killedbacterial cells.

Compositions may be presented in unit dosage form, e.g., in ampules orvials, or in multi-dose containers, with or without an addedpreservative. The composition can be a solution, a suspension, or anemulsion in oily or aqueous vehicles, and may contain formulatoryagents, such as suspending, stabilizing and/or dispersing agents. Asuitable solution is isotonic with the blood of the recipient and isillustrated by saline, Ringer's solution, and dextrose solution.Alternatively, compositions may be in lyophilized powder form, forreconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater or physiological saline. The compositions also may be in the formof a depot preparation. Such long-acting compositions may beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection.

With respect to the administration of compositions of the invention, theterms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired. In one preferredembodiment, the individual, subject, host, or patient is a human. Othersubjects may include, but are not limited to, cattle, horses, dogs,cats, guinea pigs, rabbits, rats, primates, and mice.

Administration Schedules

In general, the compositions disclosed herein may be used at appropriatedosages defined by routine testing, to obtain optimal physiologicaleffect, while minimizing any potential toxicity. The dosage regimen maybe selected in accordance with a variety of factors including age,weight, sex, medical condition of the patient; the severity of thecondition to be treated, the route of administration, and the renal andhepatic function of the patient.

Optimal precision in achieving concentrations of killed bacterial celland therapeutic within the range that yields maximum efficacy withminimal side effects may require a regimen based on the kinetics of thekilled bacterial cell and therapeutic availability to target sites andtarget cells. Distribution, equilibrium, and elimination of a killedbacterial cell or therapeutic may be considered when determining theoptimal concentration for a treatment regimen. The dosages of the killedbacterial cells and therapeutics may be adjusted when used incombination, to achieve desired effects.

Moreover, the dosage administration of the compositions may be optimizedusing a pharmacokinetic/pharmacodynamic modeling system. For example,one or more dosage regimens may be chosen and apharmacokinetic/pharmacodynamic model may be used to determine thepharmacokinetic/pharmacodynamic profile of one or more dosage regimens.Next, one of the dosage regimens for administration may be selectedwhich achieves the desired pharmacokinetic/pharmacodynamic responsebased on the particular pharmacokinetic/pharmacodynamic profile. See,e.g., WO 00/67776.

Specifically, the compositions may be administered at least once a weekover the course of several weeks. In one embodiment, the compositionsare administered at least once a week over several weeks to severalmonths.

More specifically, the compositions may be administered at least once aday for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days.Alternatively, the compositions may be administered about once everyday, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days ormore.

The compositions may alternatively be administered about once everyweek, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 weeks or more. Alternatively, the compositions maybe administered at least once a week for about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more.

Alternatively, the compositions may be administered about once everymonth, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months ormore.

The compositions may be administered in a single daily dose, or thetotal daily dosage may be administered in divided doses of two, three,or four times daily.

In method in which killed bacterial cells are administered before adrug, administration of the drug may occur anytime from several minutesto several hours after administration of the killed bacterial cells. Thedrug may alternatively be administered anytime from several hours toseveral days, possibly several weeks up to several months after thekilled bacterial cells.

More specifically, the killed bacterial cells may be administered atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23 or 24 hours before the drug. Moreover, the killedbacterial cells may be administered at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or 31 days before the administration of the drug. Inyet another embodiment, the killed bacterial cells may be administeredat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 weeks or more before the drug. In a further embodiment,the killed bacterial cells may be administered at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 months before the drug.

In another embodiment, the killed bacterial cell is administered afterthe drug. The administration of the killed bacterial cell may occuranytime from several minutes to several hours after the administrationof the drug. The killed bacterial cell may alternatively be administeredanytime from several hours to several days, possibly several weeks up toseveral months after the drug.

EXAMPLES Example 1 Killed Bacteria are Successfully Packaged with theChemotherapeutic Drug Doxorubicin

Salmonella typhimurium strain was cultured overnight in Trypticase SoyBroth (TSB). The strain was then subcultured (1:100) in 100 ml of TSBand grown to early log phase (OD₆₀₀=0.406). Bacterial count wasenumerated by plating serial dilutions on TSB agar plates and performinga colony count after overnight incubation. The result showed that theculture carried ˜5×10⁸ bacteria/ml. To kill the bacterial cells, 10 mlof the culture was incubated for 4 hrs with 500 μg/ml gentamicin and 500μg/ml chloramphenicol. A 100 μl sample was plated on TSB agar plate toascertain that the bacterial cells had been killed.

Killed bacterial cells (1×10⁹) were incubated with 60 μg/ml doxorubicinfor 2 hrs at 37° C. in 1 ml 1×BSG (buffered saline gelatin). Excess drugwas washed away from the bacterial cells by six repeat washing stepswhere the cells were centrifuged at 13,200 rpm for five minutes followedby resuspension in fresh BSG solution.

The doxorubicin was extracted from the killed bacteria following fivecycles of vortexing and sonication in the presence of 97 mMHCl-isopropyl alcohol (HCl-IPA). The samples were then diluted in anequal volume of water and the five cycles repeated. After centrifugationat 13,200 rpm for 5 min to pellet debris, the supernatants wereharvested for drug quantitation by HPLC. The mobile phase comprised 100mM ammonium formate+0.05% triethylamine (pH, 3.5):MQ:MeCN (acetonitrile)at a ratio of 28:42:30. The stationary phase comprised a LichrosphereRP18 column (MERCK) at 40° C. Detection was by excitation at 480 nm andemission at 550 nm, using a Shimadzu 10AVP system comprising anautosampler, solvent degasser, quaternary pump, column heater (40° C.)and fluorescence detector, running version 7.2 SPI rev B software(Shimadzu Corporation).

The area under the peak was interpolated in a standard curve fordoxorubicin and the results showed that ˜2 μg of doxorubicin waspackaged in 1×10⁹ killed bacterial cells.

Example 2 Tumor Regression/Stabilization Following I.V. Administrationof EGFR-Targeted, Doxorubicin-Packaged Killed Bacterial Cells in NudeMice Carrying Human Breast Cancer Xenografts

This example demonstrates that bispecific ligand-targeted andDoxorubicin-packaged intact killed bacterial cells can effect regressionof human breast cancer cell tumor xenografts established in 6 week oldfemale athymic nude mice.

As described in Example 1, killed S. typhimurium cells were packagedwith chemotherapeutic drug Doxorubicin and were purified of freeendotoxin by repeat centrifugation and washing away of the supernatant.

A bispecific antibody carrying anti-LPS and anti-human EGFRspecificities was constructed as follows. An anti-EGFR monoclonalantibody was selected because the xenografted cells were human breastcancer cells MDA-MB-468 that are known to overexpress the EGF receptoron the cell surface. A BsAb with anti-S. Typhimurium O-antigen andanti-EGFR specificities was constructed as described inPCT/US2004/041010. Briefly, bispecific antibody (BsAb) was constructedby linking an anti-S. Typhimurium O-antigen monoclonal antibody (MAb)(IgG1; Biodesign) and a MAb directed against a target cell-surfacereceptor that is mouse anti-human EGFR (IgG2a; Oncogene). The twoantibodies were cross-linked via their Fc regions using purifiedrecombinant protein A/G (Pierce Biotechnology). Briefly, protein A/G(100 μg/ml final concentration) was added to 0.5 ml of a premixedsolution containing 20 μg/ml each of anti-S. Typhimurium O-antigen andanti-human EGFR MAbs, and incubated overnight at 4° C. Excess antibodieswere removed by incubaton with protein G-conjugated magnetic beads andgentle mixing at room temperature for 40 min. After magnetic separationof the beads, the protein A/G-BsAb complex was incubated with 10⁹dox-packaged killed bacterial cells for 1 hr at room temperature to coatthem with antibody via binding of the O-antigen specific Fab arm tosurface LPS.

The mice used in this example were purchased from Animal ResourcesCentre, Perth, Wash., Australia, and all animal experiments wereperformed in compliance with the guide of care and use of laboratoryanimals, and with Animal Ethics Committee approval. The experiments wereperformed in the NSW Agriculture accredited small animal facility atEnGeneIC Pty Ltd (Sydney, NSW, Australia). Human breast adenocarcinomacells (MDA-MB-468, ATCC; human mammary epithelial cells; non-phagocytic)were grown in tissue culture to full confluency in T-75 flasks in RPMI1640 medium supplemented with 5% Bovine Calf Serum (GIBCO-BRL LifeTechnologies, Invitrogen Corporation, Carlsbad, Calif., USA) andglutamine (Invitrogen) in a humidified atmosphere of 95% air and 5% CO₂at 37° C. 1×10⁶ cells in 50 uL serum-free media together with 50 uLgrowth factor reduced matrigel (BD Biosciences, Franklin Lakes, N.J.,USA) were injected subcutaneously between the shoulder blades of eachmouse using a 23-gauge needle. The tumors were measured twice a weekusing an electronic digital caliper (Mitutoyo, Japan, precision to0.001) and mean tumor volume was calculated using the formula, length(mm)×width² (mm)×0.5=volume (mm³). 16 days post-implantation, the tumorsreached volumes between 40 mm³ and 70 mm³, and mice were randomized tofour different groups of five per group.

The experiment was designed as follows. Group 1 (control) received ani.v. dose of 100 μl of sterile physiological saline. Group 2 (control)received an i.v. dose of free Doxorubicin (7 mg/kg of mouse bodyweight). Group 3 (control) received 1×10⁸/dose of dox-packaged killedbacteria (killed S. typhimurium _(Dox)). Group 4 (experimental) received1×10⁸/dose of EGFR-targeted, dox-packaged killed bacteria (^(EGFR)killedS. typhimurium _(Dox)). All doses were administered via the i.v. routeand the doses were given on days 21, 28 and 34.

The results showed (FIG. 1) that the ^(EGFR)killed S. typhimurium _(Dox)were highly effective in achieving tumor regression/stabilization ascompared to the three controls.

Example 3 Anti-Tumor Effects Following I.V. Administration ofEGFR-Targeted, Paclitaxel-Packaged or siRNA-Kinesin SpindleProtein-Packaged Killed Bacterial Cells in Nude Mice Carrying HumanColon Cancer Xenografts

This example considers whether intact killed bacterial cells packagedwith paclitaxel or siRNA can inhibit the growth human colon cancer celltumor in vivo.

Using the methods described in Example 1, killed S. typhimurium cellswere packaged with chemotherapeutic drug paclitaxel and were purified offree endotoxin by repeat centrifugation and washing away of thesupernatant.

Separately, siRNA against the kinesin spindle protein (KSP) was packagedin the killed S. Typhimurium strain. KSP, also termed kinesin-5 or Eg5,is a microtubule motor protein that is essential for the formation ofbipolar spindles and the proper segregation of sister chromatids duringmitosis (Enos and Morris, 1990; Blangy et al., 1995; Dagenbach andEndow, 2004). Inhibition of KSP causes the formation of monopolarmitotic spindles, activates the spindle assembly checkpoint, and arrestscells at mitosis, which leads to subsequent cell death (Blangy et al.,1995, Mayer et al. 1999; Kapoor et al., 2000; Tao et al., 2005). TheKSP-siRNA double stranded oligonucleotides sequences (sense strand:5′-AAC TGG ATC GTA AGA AGG CAG-3′) were synthesized and packaged intothe killed S. Typhimurium strain by incubating 1×10¹⁰ bacteria with 1 μmof the siRNA-KSP. The co-incubation was carried out in 1× PhosphateBuffer Solution (PBS) (Gibco) for 12 hours at 37° C. with gentle mixing.Post-packaging, the bacteria were pelleted and washed twice with 1×PBSby centrifugation for 10 minutes at 16,200×g. The bacterial cells werewashed twice in 1×PBS to eliminate excess non-packaged siRNA-KSP.

A bispecific antibody carrying anti-LPS and anti-human EGFRspecificities was constructed as described in Example 2.

The mice used in this example were purchased from Animal ResourcesCentre, Perth, Wash., Australia, and all animal experiments wereperformed in compliance with the guide of care and use of laboratoryanimals, and with Animal Ethics Committee approval. The experiments wereperformed in the NSW Agriculture accredited small animal facility atEnGeneIC Pty Ltd (Sydney, NSW, Australia). Human colon cancer cells(HCT116, ATCC) were grown in tissue culture to full confluency in T-75flasks in RPMI 1640 medium supplemented with 5% Bovine Calf Serum(GIBCO-BRL Life Technologies, Invitrogen Corporation, Carlsbad, Calif.,USA) and glutamine (Invitrogen) in a humidified atmosphere of 95% airand 5% CO₂ at 37° C. 1×10⁶ cells in 50 uL serum-free media together with50 uL growth factor reduced matrigel (BD Biosciences, Franklin Lakes,N.J., USA) were injected subcutaneously between the shoulder blades inBalb/c nu/nu mice (n=8 mice per group) using a 23-gauge needle. Thetumors were measured twice a week using an electronic digital caliper(Mitutoyo, Japan, precision to 0.001), and mean tumor volume wascalculated using the formula, length (mm)×width ² (mm)×0.5=volume (mm³).16 days post-implantation, the tumors reached volumes ˜200 mm³, and micewere randomized to four different groups of eight per group.

The experiment was designed as follows. Group 1 (control) received ani.v. dose of 100 μl of sterile physiological saline. Group 2 (control)EGFR-targeted killed S. typhimurium bacteria not carrying anytherapeutic payload (G2; ^(EGFR) S. typhimurium). Group 3 (expt)EGFR-targeted killed S. typhimurium bacteria packaged withchemotherapeutic drug paclitaxel (G3; ^(EGFR) S. typhimurium_(Paclitaxel)). Group 4 (expt) EGFR-targeted killed S. typhimuriumbacteria packaged with siRNA against kinesin spindle protein (G4;^(EGFR) S. typhimurium _(siRNA-KSP)). The treatments were administeredthree times per week.

The results show (FIG. 2) that both treatments, i.e. ^(EGFR)killed S.typhimurium _(Paclitaxel) and ^(EGFR) S. typhimurium _(siRNA-KSP),showed highly significant anti-tumor effects as compared to the twocontrols. Thus, the data demonstrate that intact killed bacterial cellspackaged with paclitaxel or siRNA inhibit the growth of human coloncancer cell tumor in vivo.

Example 4 Use of Dual Treatment Comprising Receptor-Targeted KilledBacteria-Mediated shRNA Followed by Receptor-Targeted KilledBacteria-Mediated Drug Delivery

To demonstrate that receptor-targeted killed bacteria can reverse drugresistance in cancer cells in-vivo, we carried out the following studyin Balb/c nu/nu mice. For xenograft cells, we used the human coloncancer cell line Caco-2, which is highly resistant to first-linechemotherapy drugs for colon cancer, such as irinotecan and5-fluorouracil (5-FU).

Using the methods described in Example 1, S. typhimurium killed bacteriawere packaged with chemotherapeutic drug irinotecan or 5-FU. Excessirinotecan or 5-FU non-specifically attached to the outer surface of thekilled bacteria was washed away by centrifugation of the bacterial cellsat 13,200 rpm for 10 min, and the washed cells were resuspended in fresh1×PBS. This washing step was repeated.

The irinotecan or 5-FU-packaged killed S. typhimurium cells weretargeted to the EGFR via attachment of ananti-O-polysaccharide/anti-EGFR bispecific antibody to the bacterialcell surface, as described in the previous examples. An anti-EGFRmonoclonal antibody was selected because the xenograft cells, Caco-2,are known to overexpress the EGFR on the cell surface (Nyati et al.,2004). The EGFR-targeted, targeted, drug-packaged killed bacteria weredesignated ^(EGFR) S. typhimurium _(5-FU) and ^(EGFR) S. typhimurium_(Irino).

A recombinant S. typhimurium strain carrying a plasmid encodinganti-MDR1 shRNA sequence was generated as follows. The MDR-1 shRNAsequence used in this study(5′-TCGAAAGAAACCAACTGTCAGTGTAgagtactgTACACTGACAGTTGGTTTCTTTTTTT-3′) wasdescribed by Wu et al., 2003. The shRNA sequence was synthesized andsubcloned into plasmid IMG-800 (Imgenex Corp., San Diego, Calif., USA)such that the sequence could be expressed from the plasmid U6 promoter.The plasmid carries the pUC origin of replication which enables highplasmid copy numbers in bacterial cells. The recombinant plasmid wassequenced to ensure that the shRNA sequence was correct and in-frame forexpression from the U6 promoter. The recombinant plasmid was transformedinto the S. typhimurium, and the recombinant strain was designated S.typhimurium _(shRNA-MDR1). EGFR-targeted S. typhimurium _(shRNA-MDR1)was constructed by attaching the anti-O-polysaccharide/anti-EGFRbispecific antibody to the surface of the recombinant bacteria togenerate ^(EGFR) S. typhimurium _(shRNA-MDR1).

The various mice groups (five mice per group) received the followingtreatments: Group 1 (control) sterile saline; Group 2 (control) ^(EGFR)S. typhimurium _(shRNA-MDR1); Group 3 (control) EGFR-targeted,5-FU-packaged killed bacteria (^(EGFR) S. typhimurium _(5-FU)); Group 4(exp.). ^(EGFR) S. typhimurium _(shRNA-MDR1) followed by ^(EGFR) S.typhimurium _(5-FU); Group 5 (control) EGFR-targeted, Irino-packagedkilled bacteria (^(EGFR) S. typhimurium _(Irino)); Group 6 (expt)^(EGFR) S. typhimurium _(shRNA-MDR1) followed ^(EGFR) S. typhimurium_(Irino); Groups 2 to 6 received 1×109 bacterial cells, and alltreatments were i.v.

The results showed (FIG. 3) that as expected, the Caco-2 cells remainedresistant following treatments with ^(EGFR) S. typhimurium _(Irino),^(EGFR) S. typhimurium _(5-FU) and ^(EGFR) S. typhimurium _(shRNA-MDR1).Cells that received dual treatment, i.e. ^(EGFR) S. typhimurium_(shRNA-MDR1) followed by ^(EGFR) S. typhimurium _(Irino) (G6 mice) or^(EGFR) S. typhimurium _(5-FU) (G4 mice), showed highly significantreversal of drug resistance and tumor regression. The data demonstratesthat a dual treatment protocol, e.g. receptor-targeted killedbacteria-mediated shRNA delivery followed by receptor-targeted killedbacteria-mediated chemotherapeutic drug delivery, is highly effective inreversing drug resistance in non-phagocytic mammalian cells.

REFERENCES

All publications and patents mentioned in this specification areincorporated herein by reference. Reference to a publication or patent,however, does not constitute an admission as to prior art.

-   Akporiaye, E. T. & Hersh, E. Clinical aspects of intratumoral gene    therapy. Curr. Opin. Mol. Ther. 1: 443-453 (1999).-   Ambudkar, et al., Annu. Rev. Pharmacol. Toxicol. 39: 361 (1999).-   Batra R K, Wang-Johanning F, Wagner E, Garver R I Jr, Curiel D T.    Receptor-mediated gene delivery employing lectin-binding    specificity. Gene Ther. 1994 July; 1(4):255-60.-   Becker C M, Farnebo F A, lordanescu I, Behonick D J, Shih M C,    Dunning P, Christofferson R. Mulligan R C, Taylor G A, Kuo C J,    Zetter B R. Gene therapy of prostate cancer with the soluble    vascular endothelial growth factor receptor Flk1. Cancer Biol Ther.    2002 September-October; 1(5):548-53.-   Bergey's Manual of Systematic Bioloty, 2^(nd) ed., Springer-Verlag,    2001.-   Blangy, A., Lane, H. A., d'Herin, P., Harper, M., Kress, M.,    Nigg, E. A. Phosphorylation by p34cdc2 regulates spindle association    of human Eg5, a kinesin-related motor essential for bipolar spindle    formation in vivo. Cell 83: 1159-1169 (1995).-   Boucher, R. C., Pickles, R. J., Rideout, J. L., Pendergast, W. &    Yerxa, B. R. Targeted gene transfer using G protein coupled    receptors. U.S. patent application. US 2003/004123 A1. Jan. 2, 2003.-   Bullough, P. A., Hughson, F. M., Skehel, J. J., Wiley, D. C.    Structure of influenza haemagglutinin at the pH of membrane fusion.    Nature 371: 37-43 (1994).-   Caplen, Expert Opin. Biol. Ther., 3(4): 575-86 (2003).-   Caplen and Mousses, Ann. N.Y. Acad. Sci., 1002: 56-62 (2003).-   Carter, P. Improving the efficacy of antibody-based cancer    therapies. Nat Rev Cancer. 2001 November; 1(2):118-29.-   Ciliberto et al., “Cell-specific expression of a transfected human    alpha 1-antitrypsin gene,” Cell. 41: 531 (1985).-   Chen, L. M., Kaniga, K., Galan, J. E. Salmonella spp. are cytotoxic    for cultured macrophages. Mol. Microbiol. 21: 1101-1115 (1996).-   Chen, D., Murphy, B., Sung, R., Bromberg, J. S. Adaptive and innate    immune responses to gene transfer vectors: role of cytokines and    chemokines in vector function. Gene Ther, 10: 991-998 (2003).-   Clark, P. R. & Hersh, E. M. Cationic lipid-mediated gene transfer:    current concepts. Curr. Opin. Mol. Ther. 1: 158-176 (1999).-   Collins & Olive, 32 Biochem. 2795-99 (1993).-   Curiel et al., “Long-term inhibition of clinical and laboratory    human immunodeficiency virus strains in human T-cell lines    containing an HIV-regulated diphtheria toxin A chain gene,” Hum.    Gene Ther. 4: 741 (1993).-   Dagenbach, E. M., and Endow, S. A. A new kinesin tree. J. Cell Sci.    117: 3-7 (2004).-   Dang, L. H., Bettegowda, C., Huso, D. L., Kinzler, K. W.,    Vogelstein, B. Combination bacteriolytic therapy for the treatment    of experimental tumors. Proc. Natl. Acad Sci. USA 98: 15155-15160    (2001).-   de Haard, H. J. et al. A large non-immunized human Fab fragment    phage library that permits rapid isolation and kinetic analysis of    high affinity antibodies. J. Biol. Chem. 274, 18218-18230 (1999).-   de Jong, G., Telenius, A., Vanderbyl, S., Meitz, A., Drayer, J.    Efficient in-vitro transfer of a 60-Mb mammalian artificial    chromosome into murine and hamster cells using cationic lipids and    dendrimers. Chromosome Res. 9: 475-485 (2001).-   Dinges et al., “HIV-regulated diphtheria toxin A chain gene confers    long-term protection against HIV type 1 infection in the human    promonocytic cell line U937,” Hum. Gene Ther. 6: 1437 (1995).-   Dow, S. W., Fradkin, L. G., Liggitt, D. H., Willson, A. P.,    Heath, T. D., Potter, T. A. Lipid-DNA complexes induce potent    activation of innate immune responses and antitumor activity when    administered intravenously. J. Immunol. 163: 1552-1561 (1999).-   Dramsi, S. & Cossart, P. Intracellular pathogens and the actin    cytoskeleton. Annu. Rev. Cell. Dev. Biol. 14: 137-166 (1998).-   Duan et al., Mol. Cancer. Therapeutics, 3(7): 833-38 (2004).-   Dubel S, Breitling F, Kontermann R, Schmidt T, Skerra A, Little M.    Bifunctional and multimeric complexes of streptavidin fused to    single chain antibodies (scFv). J. Immunol. Methods (1995) 178,    201-209.-   Dunham, S. P. The application of nucleic acid vaccines in veterinary    medicine, Res. Vet. Sci. 73: 9-16 (2002).-   Duxbury et al., J. Am, Coll. Surg., 198: 953-59 (2004).-   El Ouahabi, A., Thiry, M., Fuks, R., Ruysschaert, J. &    Vandenbranden, M. The role of the endosome destabilizing activity in    the gene transfer process mediated by cationic lipids. FEBS Lett.    414: 187-192 (1997).-   Enos, A. P., and Morris, N. R. Mutation of a gene that encodes a    kinesin-like protein blocks nuclear division in A. nidulans. Cell    60: 1019-1027 (1990).-   Essani, K. & Dales, S. Biogenesis of vaccinia: evidence for more    than 100 polypeptides in the virion. Virology 95: 385-394 (1979).-   Farhood, H., Serbina, N. & Huang, L. The role of dioleoyl    phosphatidylethanolamine in cationic liposome mediated gene    transfer. Biochim. Biophys. Acta 1235: 289-295 (1995).-   Fasbender, A., Marshall, J., Moninger, T. O., Grunst, T., Cheng, S.    & Welsh, M. J. Effect of co-lipids in enhancing cationic    lipid-mediated gene transfer in vitro and in vivo. Gene Ther. 4:    716-725 (1997).-   Feigner, P. L., Ringold, G. M. Cationic liposome-mediated    transfection. Nature 337: 387-388 (1989).-   Ferrari, S., Griesenbach, U., Geddes, D. M., Alton, E. Immunological    hurdles to lung gene therapy. Clin Exp Immunol, 132: 1-8 (2003).-   Finlay, B. B. & Cossart, P. Exploitation of mammalian host cell    functions by bacterial pathogens. Science 276: 718-25 (1997).-   Fox, M. E., Lemmon, M. J., Mauchline, M. L, et al. Anaerobic    bacteria as a delivery system for cancer gene therapy: in vitro    activation of 5-fluorocytosine by genetically engineered clostridia.    Gene Therapy. 3: 173-178 (1996).-   Frain et al., “Binding of a liver-specific factor to the human    albumin gene promoter and enhancer,” Mol. Cell. Biol. 10: 991    (1990).-   Galan, J. E. Molecular and cellular bases of Salmonella entry into    host cells. Curr. Top. Microbiol. Immunol. 209: 43-60 (1996).-   Gao, H., Shi, W. & Freund, L. B. Mechanics of receptor-mediated    endocytosis. Proc. Natl. Acad. Sci. U.S.A. 102: 9469-9474 (2005).-   Gerlowski, L. & Jain, R. Microvascular permeability of normal and    neoplastic tissues. Microvasc. Res. 31: 288-305 (1986).-   Glennie M J, McBride H M, Worth A T, Stevenson G T. Preparation and    performance of bispecific F(ab′ gamma)2 antibody containing    thioether-linked Fab′ gamma fragments. J Immunol. 1987 Oct. 1;    139(7):2367-75.-   Gosselin M A, Lee R J. Folate receptor-targeted liposomes as vectors    for therapeutic agents. Biotechnol Annu Rev. 2002; 8:103-31-   Greber, U. F., Webster, P., Weber, J. & Helenius, A. The role of the    adenovirus protease on virus entry into cells, EMBO J. 15: 1766-1777    (1996).-   Green, N. K. & Seymour, L. W. Adenoviral vectors: systemic delivery    and tumor targeting. Cancer Gene Ther. 9: 1036-1042 (2002).-   Griffiths, A. D. et al. Isolation of high affinity human antibodies    directly from large synthetic repertoires. EMBO J. 13, 3245-3260    (1994).-   Guerrier-Takada et al., Cell, 35: 849 (1983).-   Hafez, I. M., Maurer, N. & Cullis, P. R. On the mechanism whereby    cationic lipids promote intracellular delivery of polynucleic acids.    Gene Ther. 8: 1188-1196 (2001).-   Hampel and Tritz, Biochem., 28: 4929 (1989).-   Hampel et al., Nucleic Acids Research: 299 (1990)-   Hanahan, Heritable formation of pancreatic beta-cell tumours in    transgenic mice expressing recombinant insulin/simian virus 40    oncogenes. 1985 May 9-15; 315(6015): 115-122.-   Harrison et al., “Inhibition of human immunodeficiency virus-1    production resulting from transduction with a retrovirus containing    an HIV-regulated diphtheria toxin A chain gene,” Hum. Gene Ther. 3:    461 (1992a).-   Harrison et al., “Inhibition of HIV production in cells containing    an integrated, HIV-regulated diphtheria toxin A chain gene,” AIDS    Res. Hum. Retroviruses 8: 39 (1992b).-   Hart. “Tissue specific promoters in targeting systematically    delivered gene therapy,” Semin. Oncol. 23: 154 (1996).-   Heim et al., “Wavelength mutations and posttranslational    autoxidation of green fluorescent protein,” Proc. Nat'l. Acad. Sci.    USA 91: 12501 (1994).-   Hobbs, S. K., Monsky, W. L., Yuan, F., Roberts. W. G., Griffith, L.,    Torchilin, V. P. and Jain, R. K. Regulation of transport pathways in    tumor vessels: Role of tumor type and microenvironment. Proc. Natl.    Acad. Sci. USA 95: 4607-4612 (1998).-   Hoshida T. Sunamura M, Duda D G, Egawa S, Miyazaki S, Shineha R,    Hamada H, Ohtani H, Satomi S, Matsuno S. Gene therapy for pancreatic    cancer using an adenovirus vector encoding soluble flt-1 vascular    endothelial growth factor receptor. Pancreas. 2002 August;    25(2):111-21.-   Hu, S, L Shively, A Raubitschek, M Sherman, L E Williams, J Y Wong,    J E Shively, and A M Wu. Minibody: A novel engineered    anti-carcinoembryonic antigen antibody fragment (single-chain    Fv-CH3) which exhibits rapid, high-level targeting of xenografts.    Cancer Res. 1996 56: 3055-3061.-   Hudson, P. J. & Souriau, C. Recombinant antibodies for cancer    diagnosis and therapy. Expert Opin. Biol. Ther. 1, 845-855 (2001).-   Hudson P J, Souriau C. Engineered antibodies. Nat. Med. 2003    January; 9 (1):129-34.-   Hung M C, Hortobagyi G N, Ueno N T. Development of clinical trial of    E1A gene therapy targeting HER-2/neu-overexpressing breast and    ovarian cancer. Adv Exp Med. Biol. 2000; 465:171-80.-   Jain, R. K. Transport of molecules across tumor vasculature. Cancer    Metastasis Rev. 6: 559-593 (1987).-   Jain, R. K. Delivery of molecular medicine to solid tumors. Science    271: 1079-1080 (1996).-   Jain, R. K. The Eugene M. Landis Award Lecture 1996: Delivery of    molecular and cellular medicine to solid tumors. Microcirculation 4:    1-23 (1997).-   Jain, R. K. Delivery of molecular and cellular medicine to solid    tumors. J. Control. Release 53, 49-67 (1998).-   Jain, R. K. Delivery of molecular and cellular medicine to solid    tumors. Adv. Drug Deliv. Rev. 46: 149-68 (2001).-   James, M. B., Giorgio, T. D. Nuclear-associated plasmid, but not    cell-associated plasmid, is correlated with transgene expression in    cultured mammalian cells. Mol. Ther. 1: 339-346 (2000).-   Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S. & Winter, G.    Replacing the complementarity-determining regions in a human    antibody with those from a mouse. Nature 321, 522-525 (1986).-   Kaetzel C S, Blanch V J, Hempen P M, Phillips K M, Piskurich J F,    Youngman K R The polymeric immunoglobulin receptor: structure and    synthesis. Biochem Soc Trans 25:475-480 (1997).-   Kanke, M., Sniecinski, I., & DeLuca, P. P. Interaction of    microspheres with blood constituents. I. Uptake of polystyrene    spheres by monocytes and granulocytes and effect on immune    responsiveness of lymphocytes. J. Parenter. Sci. Technol. 37:    210-217 (1983).-   Kapoor, T. M., Mayer, T. U., Coughlin, M. L., Mitchison, T. J.    Probing spindle assembly mechanisms with monastrol, a small molecule    inhibitor of the mitotic kinesin, Eg5. J. Cell Biol. 150: 975-988    (2000).-   Karpovsky B, Titus J A, Stephany D A, Segal D M. Production of    target-specific effector cells using hetero-cross-linked aggregates    containing anti-target cell and anti-Fc gamma receptor antibodies. J    Exp Med. 160:1686-701 (1984).-   Katabi et al., “Hexokinase Type II: A Novel Tumor Specific Promoter    for Gene-Targeted Therapy Differentially Expressed and Regulated in    Human Cancer Cells,” Human Gene Therapy 10: 155 (1999).-   Kelsey et al., “Species- and tissue-specific expression of human    alpha 1-antitrypsin in transgenic mice,” Genes and Devel. 1: 161    (1987).-   Kerem et al., “Identification of the cystic fibrosis gene: genetic    analysis,”Science 245: 1073 (1989).-   King, I., et al. Tumor-targeted Salmonella expressing cytosine    deaminase as an anticancer agent. Hum. Gene Ther. 13: 1225-1233    (2002).-   Kleeff J, Fukahi K, Lopez M E, Friess H. Buehler M W, Sosnowski B A,    Korc M. Targeting of suicide gene delivery in pancreatic cancer    cells via FGF receptors. Cancer Gene Ther. 2002 June; 9(6):522-32.-   Klemm, A. R. Effects of polyethylenimine on endocytosis and lysosome    stability. Biochem. Pharmacol. 56: 41-46 (1998).-   Knappik, A. et al. Fully synthetic human combinatorial antibody    libraries (HuCAL) based on modular consensus frameworks and CDRs    randomized with trinucleotides. J. Mol. Biol. 296, 57-86 (2000).-   Konerding, M. A., Miodonski, A. J., Lametschwandtner, A.    Microvascular corrosion casting in the study of tumor vascularity: a    review. Scanning Microsc. 9: 1233-1243 (1995).-   Kostelny S A, Cole M S, Tso J Y. Formation of a bispecific antibody    by the use of leucine zippers. J Immunol. 1992 Mar. 1;    148(5):1547-53.-   Kreiss, P., Cameron, 13., Rangara, R., Mailhe, P., Aguerre-Charriol,    O., Airiau, M., Scherman, D., Crouzet, J., Pitard, B. Plasmid DNA    size does not affect the physicochemical properties of lipoplexes    but modulates gene transfer efficiency. Nucleic Acids Res. 27:    3792-3798 (1999).-   Kurane et al., “Targeted Gene Transfer for Adenocarcinoma Using a    Combination of Tumor specific Antibody and Tissue-specific    Promoter,” Jpn. J. Cancer Res. 89: 1212 (1998).-   Leder et al., “Consequences of widespread deregulation of the c-myc    gene in transgenic mice: multiple neoplasms and normal development,”    Cell 45: 485 (1986).-   Lee, K-D, OhY, Portnoy, D, et al. Delivery of macromolecules into    cytosol using liposomes containing hemolysin from Listeria    monocytogenes. J. Biol. Chem. 271: 7249-7252 (1996).-   Lee, C. H., Wu, C. L., and Shiau, A. L. (2005a). Endostatin gene    therapy delivered by Salmonella choleraesuis in murine tumor    models. J. Gene Med. 6: 1382-1393.-   Lee, C. H., Wu, C. L., and Shiau, A. L. (2005b). Systemic    administration of attenuated Salmonella choleraesuis carrying    thrombospondin-1 gene leads to tumor-specific transgene expression,    delayed tumor growth and prolonged survival in the murine melanoma    model. Cancer Gene Ther. 12: 175-184.-   Lemmon, M. J., van Zijl, P., Fox, M. E., et al. Anaerobic bacteria    as a gene delivery system that is controlled by the tumor    microenvironment. Gene Therapy. 8: 791-796 (1997).-   Less, J. R., Skalak, T. C., Sevick, E. M., Jain, R. K. Microvascular    architecture in a mammary carcinoma: branching patterns and vessel    dimensions. Cancer Res. 51: 265-273 (1991).-   Less, J. R., Posner, M. C., Boucher, Y., Borochovitz, D., Wolmark,    N., Jain, R. K. Interstitial hypertension in human breast and    colorectal tumors. Cancer Res. 52: 6371-6374 (1992).-   Less, J. R., Posner, M. C., Skalak, T. C., Wolmark, N., Jain, R. K    Geometric resistance and microvascular network architecture of human    colorectal carcinoma. Microcirculation 4: 25-33 (1997).-   Li, X., Fu, G-F., Fan, Y-R., et al. Bifidobacterium adolescentis as    a delivery system of endostatin for cancer gene therapy: selective    inhibitor of angiogenesis and hypoxic tumor growth. Cancer Gene    Ther. 10: 105-111 (2003).-   Liu, Q., Muruve, D. A. Molecular basis of the inflammatory response    to adenovirus vectors. Gene Ther. 10: 935-940 (2003).-   Liu, S-C., Minton, N. P., Giaccia, A. J., Brown, J. M. Anticancer    efficacy of systemically delivered anaerobic bacteria as gene    therapy vectors targeting tumor hypoxia/necrosis. Gene Ther. 9:    291-296 (2002).-   Lorenzi, G. L., Lee, K. D. Enhanced plasmid DNA delivery using    anionic LPDII by listeriolysin O incorporation. J. Gene Med. 7:    1077-1085 (2005).-   Low K B, Ittensohn M, Le T, et al. Lipid A mutant Salmonella with    suppressed virulence and TNF□ induction retain tumor-targeting in    vivo. Nat. Biotechnol. 1999; 17: 37-41.-   Luo X, Li Z, Lin S, et al. Antitumor effect of VNP20009, an    attenuated Salmonella, in murine tumor models. Oncol Res. 2001;    12:501-508.-   MacDonald et al., “Expression of the pancreatic elastase I gene in    transgenic mice,” Hepatology 7: 425 (1987).-   Maeda, H. The enhanced permeability and retention (EPR) effect in    tumor vasculature: the key role of tumor-selective macromolecular    drug targeting. Adv. Enzyme Regul. 41: 189-207 (2001).-   Maeda, H. & Matsumura, Y. Tumoritropic and lymphotropic principles    of macromolecular drugs. Crit. Rev. Ther. Drug Carrier Syst. 6,    193-210 (1989).-   Marsh, M. & A. M. Helenius, A. M. Virus entry into animal cells.    Adv. Virus Res. 36: 107-151 (1989).-   Marshall. Carcinoembryonic antigen-based vaccines. Semin. Oncol.    2003 June; 30 (3 Suppl. 8): 30-36.-   Mason et al. “The hypogonadal mouse: reproductive functions restored    by gene therapy,” Science 234: 1372 (1986).-   Mayer, T. U., Kapoor, T. M., Haggarty, S. J., King, R. W.,    Schreiber, S. L., Mitchison, T. J. Small molecule inhibitor of    mitotic spindle bipolarity identified in a phenotype-based screen.    Science 286: 971-974 (1999).-   Menard, R., Dehio, C. & Sansonetti, P. J. Bacterial entry into    epithelial cells: the paradigm of Shigella. Trends Microbiol. 4:    220-226 (1996).-   Meyer, K., Uyechi. L. S. & Szoka, F. C. J. Manipulating the    intracellular trafficking of nucleic acids, in: K. L. Brigham (Ed.),    Gene Therapy for Diseases of the Lung, Marcel Dekker Inc, New York,    pp. 135-180 (1997).-   Minton. N. P., Mauchline, M. L., Lemmon, M. J., et al.    Chemotherapeutic tumour targeting using clostridial spores. FEMS    Microbiol. Rev. 17: 357-364 (1995).-   Monack, D. M., Raupach, B., Hromockyj, A. E., Falkow, S. Salmonella    typhimurium invasion induces apoptosis in infected macrophages.    Proc. Natl. Acad. Sci. USA. 93: 9833-9838 (1996).-   Morton & Potter, “Rhabdomyosarcoma-specific expression of the herpes    simplex virus thymidine kinase gene confers sensitivity to    ganciclovir,” J. Pharmacology & Exper. Therapeutics 286: 1066    (1998).-   Mui, B., Ahkong, Q., Chow, L. & Hope, M. Membrane perturbation and    the mechanism of lipid-mediated transfer of DNA into cells. Biochim.    Biophys. Acta 1467: 281-292 (2000).-   Nakai, T., Kanamori, T., Sando, S. & Aoyama, Y. Remarkably    size-regulated cell invasion by artificial viruses.    Saccharide-dependent self-aggregation of glycoviruses and its    consequences in glycoviral gene delivery. J. Am. Chem. Soc. 125:    8465-8475 (2003).-   Nettelbeck, D. M., Miller, D. W., Jerome, V., Zuzarte, M Watkins, S.    J., Hawkins, R. E., Muller, R. & Kontermann, R. E. Targeting of    adenovirus to endothelial cells by a bispecific single-chain diabody    directed against the adenovirus fiber knob domain and human endoglin    (CD105). Mol. Ther. 3: 882-891 (2001).-   Nieth et al., FEBS Letters, 545: 144-50 (2003).-   Nuyts S, Mellaert I V, Theys J. Landuyt W, Lambin P, Anne J.    Clostridium spores for tumor-specific drug delivery. Anti-Cancer    Drugs. 2002a; 13:115-125.-   Nuyts S, Van Mellaert L, Theys J, et al. Radio-responsive recA    promoter significantly increases TNFa production in recombinant    clostridia after 2 Gy irradiation. Gene Therapy. 2002b; 8:1197-1201.-   Ogris, M. & Wagner, E. Targeting tumors with non-viral gene delivery    systems. Drug Discovery Today 7: 479-485 (2002).-   Osaki, F., Kanamori, T., Sando, S., Sera, T. & Aoyama, Y. A quantum    dot conjugated sugar ball and its cellular uptake. On the size    effects of endocytosis in the subviral region. J. Am. Chem. Soc.    126: 6520-6521 (2004).-   Osbourn, J., Jermutus, L., Duncan, A. Current methods for the    generation of human antibodies for the treatment of autoimmune    diseases. Drug Delivery Tech 8: 845-851 (2003).-   Pack P, Pluckthun A. Miniantibodies: use of amphipathic helices to    produce functional, flexibly linked dimeric Fv fragments with high    avidity in Escherichia coli. Biochemistry. 1992 Feb. 18;    31(6):1579-84.-   Paglia P, Terrazzini N, Schulze K, Guzman C A, Colombo M P. In vivo    correction of genetic defects of monocyte/macrophages using    attenuated Salmonella as oral vectors for targeted gene delivery.    Gene Ther 2000; 7: 1725-1730.-   Pawelek J M, Low K B, Bermudes D. Tumor-targeted Salmonella as a    novel anticancer vector. Cancer Res. 1997:57:4537-4544.-   Pawelek J, Low K B, Bermudes D. Bacteria as tumourtargeting vectors.    Lancet Oncol Rev. 2003; 4:548-556.-   Perrotta and Been, Biochem., 31: 16 (1992).-   Peterson H I, Appelgren L: Tumour vessel permeability and    transcapillary exchange of large molecules of different size. Bibl    Anat 1977, 15:262-265.-   Pikaar et al., J. Infect. Dis. 172: 481 (1995).-   Pinkert et al., “An albumin enhancer located 10 kb upstream    functions along with its promoter to direct efficient,    liver-specific expression in transgenic mice,” Genes and Devel. 1:    268 (1987).-   Platt J, Sodi S, Kelley M, et al. Antitumour effects of genetically    engineered Salmonella in combination with radiation. Eur J Cancer.    2000; 36:2397-2402.-   Prasher et al., “Using GFP to see the light,” Trends in Genetics 11:    320 (1995).-   Ragheb et al., “Inhibition of human immunodeficiency virus type 1 by    Tat/Rev-regulated expression of cytosine deaminase, interferon    alpha2, or diphtheria toxin compared with inhibition by    transdominant Rev,” Hum. Gene Ther. 10: 103 (1999).-   Readhead et al., “Myelin deficient mice: expression of myelin basic    protein and generation of mice with varying levels of myelin,” Cell    48: 703 (1987).-   Ridgway J B, Presta L G, Carter P. ‘Knobs-into-holes’ engineering of    antibody CH3 domains for heavy chain heterodimerization. Protein    Eng. 1996 July; 9(7):617-21.-   Riechmann, L., Clark, M., Waldmann, H. & Winter, G. Reshaping human    antibodies for therapy. Nature 332, 323-327 (1988).-   Riezman, Trends in Cell Biology, 3: 330 (1993).-   Riordan et al., “Identification of the cystic fibrosis gene: cloning    and characterization of complementary DNA,” Science 245: 1066    (1989).-   Rommens et al., “Identification of the cystic fibrosis gene:    Chromosome walking and jumping,” Science 245: 1059 (1989).-   Rosenberg, S. A., Spiess, P. M., and Kleiner, D. E. Antitumour    effects in mice of the intravenous injection of attenuated    Salmonella typhimurium. J Immunother. 25: 218-225 (2002).-   Rossi et al., Aids Research and Human Retroviruses, 8: 183 (1992)-   Ruiz, F. E., Clancy, J. P., Perricone, M. A., Bebok, Z., Hong, J.    S., Cheng, S. H., Meeker, D. P., Young, K. R., Schoumacher, R. A.,    Weatherly, M. R., Wing, L., Morris, J. E., Sindel, L., Rosenberg,    M., van Ginkel, F. W., McGhee, J. R., Kelly, D., Lyrene, R. K.,    Sorscher, E. J. A clinical inflammatory syndrome attributable to    aerosolized lipid-DNA administration in cystic fibrosis. Hum. Gene    Ther. 12: 751-761 (2001).-   Salomon D S, Brandt R, Ciardiello F, Normanno N. Epidermal growth    factor-related peptides and their receptors in human malignancies.    Crit. Rev Oncol Hematol 1995, 19, 183-232.-   Sandvig & Deurs, Trends in Cell Biology, 4: 275 (1994).-   Saville & Collins, Cell, 61: 685-96 (1990).-   Saville & Collins, PNAS (USA), 88: 8826-30 (1991).-   Scheule, R. K. The role of CpG motifs in immunostimulation and gene    therapy. Adv. Drug Deliv. Rev. 44: 119-134 (2000).-   Seth, P., Willingham, M. C. & Pastan, I. Binding of adenovirus and    its external proteins to Triton X-114. Dependence on pH. J. Biol.    Chem. 260: 14431-14434 (1985).-   Seymour, L. W. Passive tumor targeting of soluble macromolecules and    drug conjugates. Crit. Rev. Ther. Drug Carrier Syst. 9, 135-187    (1992).-   Shangara et al., “Suicide genes: past, present and future    perspectives,” Immunology Today 21: 48 (2000).-   Shaw & Griffen, Nature 289: 409 (1981).-   Sheets, M. D. et al. Efficient construction of a large nonimmune    phage antibody library: the production of high-affinity human    single-chain antibodies to protein antigens. Proc. Natl Acad. Sci.    USA 95, 6157-6162 (1998).-   Simoes, S., Pedro, P., Duzgunes, N. & Pedrosa de Lima, M. Cationic    liposomes as gene transfer vectors: barriers to successful    application in gene therapy. Curr. Opin. Struct. Biol. 1: 147-157    (1999).-   Singh, Transferrin as a targeting ligand for liposomes and    anticancer drugs. Curr Pharm Des. 1999 June; 5(6):443-51.-   Siould, “Therapeutic siRNAs,” Trends in Pharmacological Sciences,    25(1): 22-28 (2004).-   Soghomonyan, S. A., Doubrovin, M., Pike, J., Luo, X., Ittensohn, M.,    Runyan, J. D., Balatoni, J., Finn, R., Tjuvajev, J. G., Blasberg,    R., and Bermudes, D. Positron emission tomography (PET) imaging of    tumor-localized Salmonella expressing HSV1-TK. Cancer Gene Ther. 12:    101-108 (2005).-   Sonawane, N., Szoka, F. J. & Verkman, A. Chloride accumulation and    swelling in endosomes enhances DNA transfer by polyamine-DNA    polyplexes. J. Biol. Chem. 278: 44826-44831 (2003).-   Speert et al. J. Clin. Invest., 82: 872 (1988).-   Spencer, “Developments in suicide genes for preclinical and clinical    applications,” Current Opinion in Molecular Therapeutics 2: 433-440    (2000).-   Stein, B. S., Gowda, S. D., Lifson, J. D., Penhallow, R. C.,    Bensch, K. G., Engleman, E. G. pH-independent HIV entry into    CD4-positive T cells via virus envelope fusion to the plasma    membrane, Cell 49: 659-668 (1987)-   Stockert. The asialoglycoprotein receptor: relationships between    structure, function, and expression. Physiol Rev. 1995 July;    75(3):591-609.-   Swanson, J. A. & Watts, C. Macropinocytosis. Trends Cell Biol. 5:    424-428 (1995).-   Swift et al., “Tissue-specific expression of the rat pancreatic    elastase I gene in transgenic mice,” Cell 38: 639 (1984).-   Tabata, Y., & Ikada, Y. Macrophage phagocytosis of biodegradable    microspheres composed of 1-lactic acid/glycolic acid homo- and    copolymers. J. Biomed. Mater. Res. 22: 837-858 (1988).-   Tachibana, R., Harashima, H. Ide, N., Ukitsu, S., Ohta. Y., Suzuki,    N., Kikuchi, H., Shinohara, Y., Kiwada, H. Quantitative analysis of    correlation between number of nuclear plasmids and gene expression    activity after transfection with cationic liposomes. Pharm. Res. 19:    377-381 (2002).-   Tao, W., South, V. J., Zhang, Y., Davide, J. P., Farrell, L.,    Kohl, N. E., Sepp-Lorenzino, L., Lobell, R. B. Induction of    apoptosis by an inhibitor of the mitotic kinesin KSP requires both    activation of the spindle assembly checkpoint and mitotic slippage.    Cancer Cell 8: 49-59 (2005).-   Theys, J., Landuyt, W., Nuyts, S., et al. Specific targeting of    cytosine deaminase to solid tumors by engineered Clostridium    acetobutylicum. Cancer Gene Ther. 8: 294-297 (2001).-   Thurnher M, Wagner E. Clausen H, Mechtler K, Rusconi S, Dinter A,    Birnstiel M L, Berger E G, Cotten M. Carbohydrate receptor-mediated    gene transfer to human T leukaemic cells. Glycobiology. 1994 August;    4(4):429-35.-   Todorovska, A. et al. Design and application of diabodies,    triabodies and tetrabodies for cancer targeting. J. Immunol. Methods    248, 47-66 (2001).-   Tomlinson, I. & Holliger, P. Methods for generating multivalent and    bispecific antibody fragments. Methods Enzymol. 326, 461-479 (2000).-   Vaughan, T. J. et al. Human antibodies with subnanomolar affinities    isolated from a large non-immunized phage display library. Nature    Biotechnol. 14, 309-314 (1996).-   Vaughan, T. J., Osbourn, J. K. & Tempest, P. R. Human antibodies by    design. Nature Biotechnol. 16, 535-539 (1998).-   Verhoeyen, M., Milstein, C. & Winter, G. Reshaping human antibodies:    grafting an antilysozyme activity. Science 239, 1534-1536 (1988).-   Wakimoto, H., Johnson, P. R., Knipe, D. M., Chiocca, E. A. (2003)    Effects of innate immunity on herpes simplex virus and its ability    to kill tumor cells. Gene Ther. 10: 983-990 (2003).-   Warren, B. A. The vascular morphology of tumors. Tumor Blood    Circulation: Angiogenesis, Vascular Morphology and Blood Flow of    Experimental and Human Tumors. Edited by Peterson H-1. Boca Raton,    CRC Press, Inc., pp 1-48 (1979).-   Wattiaux, R., Laurent, N., Wattiaux-De Coninck, S. & Jadot, M.    Endosomes, lysosomes: their implication in gene transfer. Adv. Drug    Deliv. Rev. 41: 201-208 (2000).-   Weiss. S. & Chakraborty, T. Transfer of eukaryotic expression    plasmids to mammalian host cells by bacterial carriers. Curr. Opin.    Biotechnol. 12: 467-472 (2001).-   Whitmore, M., Li, S., Huang, L. LPD lipopolyplex initiates a potent    cytokine response and inhibits tumor growth. Gene Ther. 6: 1867-1875    (1999).-   Whitmore, M. M., Li, S., Falo, L., Jr, Huang, L. Systemic    administration of LPD prepared with CpG oligonucleotides inhibits    the growth of established pulmonary metastases by stimulating innate    and acquired antitumor immune responses. Cancer Immunol. Immunother.    50: 503-514 (2001).-   Wickham, T. J., Segal, D. M., Roelvink, P. W., Carrion, M. E.,    Lizonova, A., Lee, G. M. & Kovesdi, I. Targeted adenovirus gene    transfer to endothelial and smooth muscle cells by using bispecific    antibodies. J. Virol. 70: 6831-6838 (1996).-   Wright & Jong, Experimental Medi., 163: 1245 (1986).-   Wrobel, I. & Collins, D. Fusion of cationic liposomes with mammalian    cells occurs after endocytosis. Biochim. Biophys. Acta 1235: 296-304    (1995).-   Wu, et al., Cancer Res., 63: 1515-19 (2003).-   Xu, Y. & Szoka, F. C. Mechanism of DNA release from cationic    liposome/DNA complexes used in cell transfection. Biochem. J. 35:    5616-5623 (1996).-   Yague et al., Gene Therapy, 11: 1170-74 (2004).-   Yamada, H., Matsumoto, S., Matsumoto, T., Yamada, T., and    Yamashita, U. Murine IL-2 secreting recombinant Bacillus    Calmette-Guerin augments macrophage mediated cytotoxicity against    murine bladder cancer MBT-2. J. Urol. 164: 526-531 (2000).-   Yazawa K, Fujimori M, Amano J, Kano Y, Taniguchi S. Bifidobacterium    longum as a delivery system for cancer gene therapy: Selective    localization and growth in hypoxic tumors. Cancer Gene Ther. 2000;    7:269-274.-   Yazawa K, Fujimori M, Nakamura T, et al. Bifidobacterium longum as a    delivery system for cancer gene therapy of chemically induced rat    mammary tumors. Breast Cancer Res Treat. 2001; 66:165-170.-   Yazawa et al., “Current progress in suicide gene therapy for    cancer,” World J. Surg. 26: 783 (2002).-   Yew, N. S., Wang, K. X., Przybylska, M., Bagley, R. G., Stedman, M.,    Marshall, J., Scheule, R. K., Cheng, S. H. Contribution of plasmid    DNA to inflammation in the lung after administration of cationic    lipid:pDNA complexes. Hum. Gene Ther. 10: 223-234 (1999).-   Yu, Y. A., Shabahang, S., Timiryasova, T. M., et al. Visualization    of tumors and metastases in live animals with bacteria and vaccinia    virus encoding light-emitting proteins. Nat. Biotechnol. 22: 313-320    (2004).-   Yuan, F., Leunig, M., Huang, S. K., Berk, D. A., Papahadjopoulos, D.    & Jain, R. K. Microvascular permeability and interstitial    penetration of sterically stabilized (stealth) liposomes in a human    tumor xenograft. Cancer Res. 54, 3352-3356 (1994).-   Yuan, F., Dellian, M., Fukumura, D., Leunig, M., Berk, D.,    Torchillin, V. & Jain, R. Vascular permeability in a human tumor    xenograft: molecular size dependence and cutoff size. Cancer Res.    55: 3752-3756 (1995).-   Yuhua, L., Kunyuan, G., Hui, C., et al. Oral cytokine gene therapy    against murine tumor using attenuated Salmonella typhimurium.    Int. J. Cancer 94: 438-443 (2001).-   Zelphati, O. & Szoka, F. Intracellular distribution and mechanism of    delivery of oligonucleotides mediated by cationic lipids. Pharm.    Res. 13: 1367-1372 (1996).-   Zelphati, 0. & Szoka, F. Mechanism of oligonucleotide release from    cationic liposomes. Proc. Natl. Acad. Sci. U.S.A. 93: 11493-11498    (1996).-   Zhao, Y., Zhu, L., Lee, S., Li, L., Chang, E., Soong, N. W.,    Douer, D. & Anderson. W. F. Identification of the block in targeted    retroviral-mediated gene transfer. Proc. Natl. Acad. Sci. USA 96:    4005-4010 (1999).-   Zhou, X., Mantis, N., Zhang, X. R., Potoka, D. A., Watkins, S. C.,    and Ford, H. R. Salmonella typhimurium induces apoptosis in human    monocyte-derived macrophages. Microbiol. Immunol. 44: 987-995    (2000).-   Ziady A G, Perales J C, Ferkol T, Gerken T, Beegen H, Perlmutter D    H, Davis P B. Gene transfer into hepatoma cell lines via the serpin    enzyme complex receptor. Am J Physiol. 1997 August; 273(2 Pt    1):G545-52.-   WO 81/01145-   WO 88/07378-   WO 95/21191-   WO 00/67776-   U.S. Pat. No. 4,975,278-   U.S. Pat. No. 4,987,071

We claim:
 1. A delivery method that comprises bringing a compositioncomprising a plurality of intact killed bacterial cells comprising abioactive agent selected from the group consisting of a functionalnucleic acid that directly interacts with a transcript encoding aprotein and a drug not comprising a nucleic acid encoding a drug,wherein the plurality of intact killed bacterial cells comprises anadhesin that binds to a non-phagocytic mammalian cell surface receptor,into contact with endocytosis-competent mammalian cells comprising thenon-phagocytic mammalian cell surface receptor, such that the intactkilled bacterial cells are endocytosed by the mammalian cells byreceptor-mediated endocytosis, thereby delivering the bioactive agent tothe mammalian cells.
 2. The method of claim 1, wherein the pluralitycomprises a therapeutically significant concentration of the bioactiveagent.
 3. The method of claim 1, wherein the contact is in vivo.
 4. Themethod of claim 1, wherein the contact is in vitro.
 5. The method ofclaim 1, wherein the bioactive agent is the functional nucleic acid. 6.The method of claim 1, wherein the functional nucleic acid targets thetranscript of a protein that contributes to resistance to the drug. 7.The method of claim 1, wherein the bioactive agent is the drug.
 8. Themethod of claim 7, wherein the drug is a small molecule drug.
 9. Themethod of claim 7, wherein the drug is a cancer chemotherapeutic agent.10. The method of claim 1, wherein the composition comprising theplurality of intact killed bacterial cells further comprises abispecific ligand comprised of a first arm that carries specificity fora surface structure of a bacterial cell of (i) and a second arm thatcarries specificity for a non-phagocytic mammalian cell surfacereceptor.
 11. The method of claim 10, wherein the bispecific ligandcomprises an antibody or antibody fragment.
 12. The method of claim 10,wherein the bispecific ligand comprises a polypeptide or carbohydrate.13. The method of claim 10, wherein the surface structure of a bacterialcell is an O-polysaccharide component of a lipopolysaccharide on thekilled bacterial cell surface.
 14. The method of claim 10, wherein thefirst arm and the second arm are monospecific.
 15. The method of claim10, wherein the first arm and the second arm are multivalent.